<PMCID>2216744</PMCID>
			<div type="paragraph">
				<div type="sentence">
					To obtaina beta-arrestin2mutant that is not ubiquitinatedupon 7TMRstimulation, we madeconservative changes of groups of lysines to arginines, overexpressedFLAG-taggedmutants in COS-7cells and tested the beta-arrestin precipitates for the ubiquitination signal inducedby one-minuteisoproterenolstimulation(Fig 1 A, B).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(TMR)</desc>
					<desc type="relation">modifiedForm(TMR_inactive, TMR_active)</desc>
					<desc type="action">reactionModulationMediationInit(beta-arrestin2, TMR_inactive, TMR_active, noEffect, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">gene(made)</desc>
					<desc type="ontological">domain(FLAG-tagged, unknownProtein, unknownDefined)</desc>
					<desc type="ontological">cell(COS-7)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="relation">modifiedForm(isoproterenol_inactive, isoproterenol_active)</desc>
					<desc type="action">reactionModulationMediationInit(COS-7, isoproterenol_inactive, isoproterenol_active, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="reference">referenceFigure(Fig 1 A, B)</desc>
				</div>
				<div type="sentence">
					Surprisingly, elimination of such a signal required replacement of all of beta-arrestin2's31 lysineresidues(mutant beta-arrestin2 ^0KFig 1B).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">molecule(lysine)</desc>
					<desc type="ontological">molecule(lysine)</desc>
					<desc type="ontological">domain(residues, unknownProtein, unknownDefined)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="reference">referenceFigure(Fig 1B)</desc>
				</div>
				<div type="sentence">
					When a FLAGepitope-tagged beta-arrestin2 ^0Kis over expressedin HEK-293cells, no ubiquitination smear is detectedupon isoproterenolstimulation(Fig 1C).
					<desc type="ontological">gene(FLAG)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">cell(HEK-293)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="relation">modifiedForm(isoproterenol_inactive, isoproterenol_active)</desc>
					<desc type="action">reactionModulationMediationInit(HEK-293, isoproterenol_inactive, isoproterenol_active, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="reference">referenceFigure(Fig 1C)</desc>
				</div>
				<div type="sentence">
					Although these experiments indicatethat beta-arrestin2 ^0Kcan be expressedas a properly folded protein that is isolated and detectedby the epitope tag, a concern still remains whether beta-arrestin2 ^0Kdespite its 31 lysineto argininechanges is a bona fide form of beta-arrestin.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">gene(tag)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">molecule(lysine)</desc>
					<desc type="ontological">molecule(lysine)</desc>
					<desc type="ontological">molecule(arginine)</desc>
					<desc type="ontological">molecule(arginine)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					To test whether the basic folding and binding properties of beta-arrestin2 ^0Kare retained, we compared the binding of in vitrotranslatedbeta-arrestin2and beta-arrestin2 ^0Kto purified recombinant beta2ARreconstituted in vesicles.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">method(in vitro)</desc>
					<desc type="ontological">process(translated)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
				</div>
				<div type="sentence">
					We also tested beta-arrestin2-Ub for receptor binding under the same conditions.
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					In these in vitroassays, both WTand beta-arrestin2 ^0Krepresent non-ubiquitinatedforms and only beta-arrestin2-Ub chimera constitutes the ubiquitinatedform.
					<desc type="ontological">method(in vitro)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="action">quantityModulationInit(beta-arrestin2 ^0K, beta-arrestin2, increase, unknownDistance, confirmed)</desc>
				</div>
				<div type="sentence">
					As shownin Fig 2A and B, beta-arrestin2 ^0Kbound to the beta2ARto the same extent as beta-arrestin2.
					<desc type="reference">referenceFigure(Fig 2A and B)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					However the presence of a single ubiquitin moiety increasedthe binding by fourfold (Fig 2A and B).
					<desc type="reference">referenceFigure(Fig 2A and B)</desc>
				</div>
				<div type="sentence">
					These experiments suggest that while both non-ubiquitinatedforms of beta-arrestin2(i.e.
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					WTand 0K) are equipotent for beta2ARbinding, there is more binding between the beta2ARand the ubiquitinatedform (i.e.
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
				</div>
				<div type="sentence">
					beta-arrestin2-Ub).
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					When binding was performed in the presence of isoproterenol, a small increasewas observedfor all three beta-arrestin forms (data not shown).
					<desc type="ontological">molecule(isoproterenol)</desc>
				</div>
				<div type="sentence">
					We hypothesized that reconstituted beta2ARwas already in an activatedconformation due to the presence of zinterol in purification buffers.
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
				</div>
				<div type="sentence">
					If so, inclusion of an antagonist could alter the observedbinding.
				</div>
				<div type="sentence">
					When beta-arrestin-receptor complex formation was tested in the presence of propranolol, we founda dramatic decreasein binding for all three beta-arrestin forms (Fig 2A and B), suggesting that propranololdestabilizes but does not eliminate receptor-beta-arrestin binding in these experiments.
					<desc type="ontological">molecule(propranolol)</desc>
					<desc type="reference">referenceFigure(Fig 2A and B)</desc>
					<desc type="ontological">molecule(propranolol)</desc>
				</div>
				<div type="sentence">
					Moreover, when reconstituted receptor samples were probed with a beta2ARspecific phosphoserine antibody (serines 355,356), a small amount of phosphorylation was detected(Fig 2Cupper panel).
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="reference">referenceFigure(Fig 2C)</desc>
				</div>
				<div type="sentence">
					Addition of GRK2leads to an increasein the phosphorylation signal and isoproterenolaugments it further (Fig 2C).
					<desc type="ontological">gene(GRK2)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="reference">referenceFigure(Fig 2C)</desc>
				</div>
				<div type="sentence">
					We observeda comparable increasein binding above basal conditions for all three beta-arrestin forms upon GRK2phosphorylation and isoproterenoltreatmentof the reconstituted beta2AR(Fig 2D).
					<desc type="ontological">gene(GRK2)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">method(treatment)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="reference">referenceFigure(Fig 2D)</desc>
				</div>
				<div type="sentence">
					Collectively, these in vitrobinding assays confirmthat although ubiquitinatedbeta-arrestin2forms a tight complex with the beta2AR, nonubiquitinated beta-arrestin2can bind reconstituted beta2ARand that the protein-protein interaction domain(s) between the receptor and beta-arrestin2 ^0Kis mostly unperturbed.
					<desc type="ontological">method(in vitro)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					To determine the isoproterenol-stimulatedbinding of beta-arrestin2 ^0Kto the beta2ARin a cellular context we employed immunoprecipitationassays utilizing chemical crosslinking with a sufhydryl-reactive compound, DTME (Fig 3).
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="action">quantityModulationInit(isoproterenol, beta-arrestin2 ^0K, increase, unknownDistance, confirmed)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">method(immunoprecipitation)</desc>
					<desc type="reference">referenceFigure(Fig 3)</desc>
				</div>
				<div type="sentence">
					We used COS-7cells transiently transfectedwith FLAG-beta2ARand beta-arrestin2 ^0K-GFP, immunoprecipitatedthe receptors under nonstimulated or stimulatedconditions (5 min, 1 μMisoproterenol) and detectedbeta-arrestin2 ^0K-GFPby Western blotting(Fig 3A).
					<desc type="ontological">cell(COS-7)</desc>
					<desc type="ontological">method(transfected)</desc>
					<desc type="ontological">gene(FLAG)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="relation">complex(FLAG-beta2)</desc>
					<desc type="relation">complexElement(FLAG, FLAG-beta2)</desc>
					<desc type="relation">complexElement(beta2, FLAG-beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2 ^0K, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="ontological">method(immunoprecipitated)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2 ^0K, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="action">quantityModulationInit(isoproterenol, beta-arrestin2 ^0K-GFP, increase, unknownDistance, confirmed)</desc>
					<desc type="ontological">method(blotting)</desc>
					<desc type="reference">referenceFigure(Fig 3A)</desc>
				</div>
				<div type="sentence">
					beta-arrestin2 ^0K-GFPbinds to activatedreceptors with a 2-3 fold agonist-inducedrecruitment(Fig 3B).
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2 ^0K, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="ontological">process(recruitment)</desc>
					<desc type="reference">referenceFigure(Fig 3B)</desc>
				</div>
				<div type="sentence">
					In similar assays, the WTand beta-arrestin2-Ub were recruited10-12 and 12-15 fold, respectively (data not shown).
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					These experiments further suggest that beta-arrestin2 ^0K-GFPalbeit much weaker than the WTnevertheless binds the beta2ARupon agonist stimulation.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2 ^0K, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
				</div>
				<div type="sentence">
					Likely, the robust association of beta-arrestin2 ^0Kand the beta2ARdoes not occurin cells due to a lackof beta-arrestin2ubiquitination, which helps to stabilize the complex.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">gene(lack)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(beta-arrestin2)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					We next transfectedHEK-293cells stably expressingthe beta2ARwith either beta-arrestin2-GFP, GFP-beta-arrestin2-Ub (stable ubiquitination), or beta-arrestin2 ^0K-GFP(no ubiquitination) and examined the translocation patterns inducedby isoproterenol.
					<desc type="ontological">method(transfected)</desc>
					<desc type="ontological">cell(HEK-293)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2, beta-arrestin2-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2-GFP)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="relation">complex(GFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(GFP, GFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(beta-arrestin2, GFP-beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2 ^0K, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="action">associationModulationInit(GFP-beta-arrestin2, beta-arrestin2 ^0K-GFP, isoproterenol, unknownparticle, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					All three beta-arrestin variants are uniformly distributed in the cytosolprior to agonist treatment(Fig 4 A-C).
					<desc type="ontological">compartment(cytosol)</desc>
					<desc type="ontological">method(treatment)</desc>
					<desc type="reference">referenceFigure(Fig 4 A-C)</desc>
				</div>
				<div type="sentence">
					Within one-minuteof agonist-stimulation, both WTand GFP-beta-arrestin2-Ub are recruitedto the cell membraneand form distinct puncta and at 30 minutesGFP-beta-arrestin2-Ub is recruitedto endosomalvesicles (Fig 4B), while the WTremains at the membrane(Fig 4A).
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="relation">complex(GFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(GFP, GFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(beta-arrestin2, GFP-beta-arrestin2)</desc>
					<desc type="ontological">compartment(cell membrane)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="relation">complex(GFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(GFP, GFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(beta-arrestin2, GFP-beta-arrestin2)</desc>
					<desc type="ontological">compartment(endosomal)</desc>
					<desc type="reference">referenceFigure(Fig 4B)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="reference">referenceFigure(Fig 4A)</desc>
				</div>
				<div type="sentence">
					As previously shown, with a 'Class A'receptora stably ubiquitinatedbeta-arrestin traffics into endosomeswhile the transiently ubiquitinatedWT beta-arrestin dissociates and remains at the plasma membrane.
					<desc type="ontological">gene(A')</desc>
					<desc type="ontological">protein(A' receptor)</desc>
					<desc type="ontological">compartment(endosomes)</desc>
					<desc type="ontological">method(WT beta)</desc>
					<desc type="ontological">compartment(plasma membrane)</desc>
				</div>
				<div type="sentence">
					On the other hand, agonist stimulation for 1 or 30 mindoes not lead to a major changein the intracellulardistribution of beta-arrestin2 ^0K-GFP(Fig 4C, center panels).
					<desc type="ontological">compartment(intracellular)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2 ^0K, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="reference">referenceFigure(Fig 4C)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					To test whether the loss of translocation correlates with a loss of ubiquitination owing to cumulative lysinemutations, we examined isoproterenol-inducedrecruitmentof all the mutants shownin Fig 1A, B.
					<desc type="ontological">molecule(lysine)</desc>
					<desc type="ontological">molecule(lysine)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">process(recruitment)</desc>
					<desc type="reference">referenceFigure(Fig 1A, B)</desc>
				</div>
				<div type="sentence">
					When the GFP-tagged version of each mutant was coexpressed with HA-beta2ARin HEK-293cells, we observednormal cytosolicexpressionunder basal conditions for all the beta-arrestin2variants (supplementary Fig 1).
					<desc type="ontological">protein(GFP)</desc>
					<desc type="ontological">proteinOrGene(HA)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="relation">complex(HA-beta2)</desc>
					<desc type="relation">complexElement(HA, HA-beta2)</desc>
					<desc type="relation">complexElement(beta2, HA-beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="ontological">cell(HEK-293)</desc>
					<desc type="ontological">protein(cytosolic)</desc>
					<desc type="ontological">gene(cytosolic)</desc>
					<desc type="ontological">process(expression)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="reference">referenceFigure(supplementary Fig 1)</desc>
				</div>
				<div type="sentence">
					However, upon 1 minisoproterenolstimulation, a decreasein the levelof recruitmentwas observedcorrelating with the ubiquitination status of beta-arrestin2(Fig 5middle panels and Fig 1B).
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="relation">modifiedForm(isoproterenol_inactive, isoproterenol_active)</desc>
					<desc type="ontological">process(recruitment)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="reference">referenceFigure(Fig 5)</desc>
					<desc type="reference">referenceFigure(Fig 1B)</desc>
				</div>
				<div type="sentence">
					Some amount of recruitmentremains even when 26 lysineresiduesare altered.
					<desc type="ontological">process(recruitment)</desc>
					<desc type="ontological">molecule(lysine)</desc>
					<desc type="ontological">molecule(lysine)</desc>
					<desc type="ontological">domain(residues, unknownProtein, unknownDefined)</desc>
				</div>
				<div type="sentence">
					The only mutant that is totally defective in translocation is beta-arrestin2 ^0Kwhere no ubiquitination sites remain.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
				</div>
				<div type="sentence">
					These data suggest a strong correlation between beta-arrestin ubiquitination status and its ability to bind activatedreceptors at the plasma membrane.
					<desc type="ontological">compartment(plasma membrane)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					The above experiments also suggest that eliminating beta-arrestin ubiquitination decreasesits binding affinity for receptors in vivo and hence only unstable receptor-beta-arrestin2 ^0K-GFPcomplexes arise at the cell membrane.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2 ^0K, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="ontological">compartment(cell membrane)</desc>
					<desc type="action">localizationInit(beta-arrestin2 ^0K-GFP, cell membrane, hypothesis, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					In the caseof 'Class A'receptors, such as the beta2AR, although receptor-beta-arrestin complexes are initially formed at the plasma membrane, these complexes are not long-lived.
					<desc type="ontological">gene(case)</desc>
					<desc type="ontological">gene(A')</desc>
					<desc type="ontological">protein(A' receptors)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">compartment(plasma membrane)</desc>
					<desc type="action">transportModulationPerturbatorInit(case, A' receptors, beta2-AR, unknownLocalization, plasma membrane, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					Thus, beta-arrestin is rapidly deubiquitinated, dissociates from the receptor, and the receptor alone traffics into endosomes.
					<desc type="ontological">compartment(endosomes)</desc>
				</div>
				<div type="sentence">
					This indicatesthat events occurring during pit/vesicle formation (e.g.
					<desc type="ontological">gene(pit)</desc>
				</div>
				<div type="sentence">
					beta-arrestin deubiquitination) can influence the stability of beta-arrestin-receptor complexes.
				</div>
				<div type="sentence">
					Possibly, the lackof ubiquitin moieties on beta-arrestin2 ^0K-GFPleads to its rapid disengagement from the receptor complex.
					<desc type="ontological">gene(lack)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2 ^0K, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2 ^0K-GFP)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					We hypothesized that beta-arrestin2 ^0Kbinds activatedreceptor at the plasma membranebut that its deficiency in ubiquitination resultsin decreasedstability of the complex as the receptor moves into pits.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">compartment(plasma membrane)</desc>
				</div>
				<div type="sentence">
					If this were true, then blockingthe internalization of receptors should resultin the retention of beta-arrestin2 ^0K-receptor complexes at the plasma membrane.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">compartment(plasma membrane)</desc>
					<desc type="action">localizationInit(beta-arrestin2 ^0K, plasma membrane, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					Indeed, when we inhibitedthe internalization of either the beta2AR(Fig 6A) or the 'Class B'V2R(Fig 6B) by co-expressing dynamin^K44A (a classical inhibitor of endocytosis, (19,20)) we trapped activatedreceptors as well as beta-arrestin2 ^0K-GFPat the membrane.
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="reference">referenceFigure(Fig 6A)</desc>
					<desc type="ontological">gene(B')</desc>
					<desc type="ontological">protein(B')</desc>
					<desc type="ontological">protein(V2R)</desc>
					<desc type="reference">referenceFigure(Fig 6B)</desc>
					<desc type="ontological">gene(co)</desc>
					<desc type="ontological">process(endocytosis)</desc>
					<desc type="ontological">inhibitorAgainst(co, endocytosis)</desc>
					<desc type="reference">reference(19)</desc>
					<desc type="reference">reference(20)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2 ^0K, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="action">localizationInit(beta-arrestin2 ^0K-GFP, membrane, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					These experiments clearly indicatethat the deficiency in ubiquitination does not inhibittranslocation of cytosolicbeta-arrestin2 ^0Kto activatedreceptors at the cell membrane, but rather decreasesthe stability of the receptor-beta-arrestin complexes that are formed.
					<desc type="ontological">protein(cytosolic)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">compartment(cell membrane)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					A characteristic feature of beta-arrestin2is its ability to augment receptor internalization upon over-expression(9).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">process(expression)</desc>
					<desc type="reference">reference(9)</desc>
				</div>
				<div type="sentence">
					This effect is particularly striking in COS-7cells, which expressvery low levelsof endogenous beta-arrestin2(21).
					<desc type="ontological">cell(COS-7)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="reference">reference(21)</desc>
				</div>
				<div type="sentence">
					To characterize the ability of beta-arrestin2 ^0Kto promotereceptor internalization, we over-expressedit together with HA-taggedbeta2ARor V2Rand measured the decreasein cell surfacereceptors after a 30 minagonist treatment.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">domain(HA-tagged, unknownProtein, unknownDefined)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">protein(V2R)</desc>
					<desc type="ontological">compartment(cell surface)</desc>
					<desc type="ontological">method(treatment)</desc>
				</div>
				<div type="sentence">
					Over expressionof beta-arrestin2 ^0Kdoes not lead to any increasein receptor internalization whereas WTbeta-arrestin2leads to an approximately 2.5-fold increasein both beta2ARand V2Rinternalization (Fig 7 A, B).
					<desc type="ontological">process(expression)</desc>
					<desc type="ontological">gene(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="action">quantityModulationInit(beta-arrestin2, beta2-AR, increase, unknownDistance, confirmed)</desc>
					<desc type="ontological">protein(V2R)</desc>
					<desc type="reference">referenceFigure(Fig 7 A, B)</desc>
				</div>
				<div type="sentence">
					Similarly, over-expressionof beta-arrestin2 ^0Kresultsin no changein receptor internalization in HEK-293cells (Fig 7 C, D).
					<desc type="ontological">process(expression)</desc>
					<desc type="ontological">gene(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">cell(HEK-293)</desc>
					<desc type="reference">referenceFigure(Fig 7 C, D)</desc>
				</div>
				<div type="sentence">
					Predictably, because of the unstable interaction of beta-arrestin2 ^0Kwith activatedreceptors than the WT, the mutant does not have any inhibitory effect on receptor internalization in both cell types.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">method(WT)</desc>
				</div>
				<div type="sentence">
					We have previously demonstratedthat the stably ubiquitinatedform of beta-arrestin2(beta-arrestin2-Ub) enhancesreceptor internalization compared to the WT(13).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="reference">reference(13)</desc>
				</div>
				<div type="sentence">
					In contrast, beta-arrestin2 ^0K, which is not ubiquitinated, forms unstable complexes with activatedreceptors, does not support internalization of either the beta2ARor the V2R.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">protein(V2R)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					We further tested if the above lackof effect of beta-arrestin2 ^0Kto enhancereceptor internalization was due to altered binding to endocyticproteins such as clathrin and AP-2.
					<desc type="ontological">gene(lack)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">process(endocytic)</desc>
					<desc type="ontological">protein(AP-2)</desc>
				</div>
				<div type="sentence">
					Clathrin binds beta-arrestin directly and stoichiometrically and beta-arrestin-clathrin binding is essential for receptor internalization via clathrin-coated vesicles (7).
					<desc type="reference">reference(7)</desc>
				</div>
				<div type="sentence">
					AP-2 -beta-arrestin interaction is required for the movement of receptors to clathrin-coated pits (8).
					<desc type="ontological">protein(AP-2 -beta)</desc>
					<desc type="reference">reference(8)</desc>
				</div>
				<div type="sentence">
					When beta-arrestin2, beta-arrestin2 ^0Kor beta-arrestin2-Ub were immunoprecipitatedfrom COS-7cells transfectedwith HA-beta2ARafter 0, 1 and 10 minutesof isoproterenoltreatment, an agonist-dependent increasein clathrin binding was observedfor both WTand beta-arrestin2-Ub but not for beta-arrestin2 ^0K(Fig 8A, B).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">method(immunoprecipitated)</desc>
					<desc type="ontological">cell(COS-7)</desc>
					<desc type="ontological">method(transfected)</desc>
					<desc type="ontological">proteinOrGene(HA)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="relation">complex(HA-beta2)</desc>
					<desc type="relation">complexElement(HA, HA-beta2)</desc>
					<desc type="relation">complexElement(beta2, HA-beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">method(treatment)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="reference">referenceFigure(Fig 8A, B)</desc>
				</div>
				<div type="sentence">
					beta-arrestin2 ^0Kdisplayed only a weak interaction and a decreasein binding in the presence of isoproterenol(Fig 8A, B).
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="action">quantityModulationInit(beta-arrestin2 ^0K, isoproterenol, decrease, unknownDistance, confirmed)</desc>
					<desc type="reference">referenceFigure(Fig 8A, B)</desc>
				</div>
				<div type="sentence">
					For the wild type beta-arrestin2, we founda five-fold increasein AP-2binding at 1 minof agonist treatmentand this binding decreasedto basal levelsat 10 min(Fig 8A, C).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(AP-2)</desc>
					<desc type="ontological">method(treatment)</desc>
					<desc type="reference">referenceFigure(Fig 8A, C)</desc>
				</div>
				<div type="sentence">
					A similar time courseof AP-2-beta-arrestin2interaction has been previously reported (8).
					<desc type="ontological">protein(AP-2)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="relation">complex(AP-2-beta-arrestin2)</desc>
					<desc type="relation">complexElement(AP-2, AP-2-beta-arrestin2)</desc>
					<desc type="relation">complexElement(beta-arrestin2, AP-2-beta-arrestin2)</desc>
					<desc type="reference">reference(8)</desc>
				</div>
				<div type="sentence">
					Surprisingly, both beta-arrestin2-Ub and beta-arrestin2 ^0Kdisplayed robust binding to AP-2under both basal and stimulatedconditions (Fig 8A and 8C).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(AP-2)</desc>
					<desc type="reference">referenceFigure(Fig 8A and 8C)</desc>
				</div>
				<div type="sentence">
					Understandably, beta-arrestin2 ^0K'sweak interaction with clathrin upon isoproterenolstimulationcould be a major factor in its inability to promotereceptor endocytosis.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="relation">modifiedForm(isoproterenol_inactive, isoproterenol_active)</desc>
					<desc type="ontological">process(endocytosis)</desc>
				</div>
				<div type="sentence">
					Unlike the previously reported beta-arrestin1^319-418 which binds clathrin but lacks receptor interaction (22), beta-arrestin2 ^0Kdid not act as an inhibitor of receptor internalization.
					<desc type="reference">reference(22)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					Previous studies have shownthat beta-arrestin-Rafcomplexes are stable since their isolation is possible by gel filtrationas well as by coimmunoprecipitation(18,23).
					<desc type="ontological">gene(Raf)</desc>
					<desc type="ontological">method(filtration)</desc>
					<desc type="ontological">method(coimmunoprecipitation)</desc>
					<desc type="reference">reference(18)</desc>
					<desc type="reference">reference(23)</desc>
				</div>
				<div type="sentence">
					We tested the interaction of the above three beta-arrestin forms with the MAPKKK, cRaf(Fig 9A) and did not observeany differences between the WTand beta-arrestin2 ^0Kin their ability to bind myc-c-Raf 1.
					<desc type="ontological">gene(cRaf)</desc>
					<desc type="reference">referenceFigure(Fig 9A)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">peptide(myc)</desc>
					<desc type="ontological">protein(c-Raf 1)</desc>
					<desc type="relation">complex(myc-c-Raf 1)</desc>
					<desc type="relation">complexElement(myc, myc-c-Raf 1)</desc>
					<desc type="relation">complexElement(c-Raf 1, myc-c-Raf 1)</desc>
				</div>
				<div type="sentence">
					In these assays, beta-arrestin2-Ub, however, bound more c-Rafthan the WT(Fig 9A).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(c-Raf)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="reference">referenceFigure(Fig 9A)</desc>
				</div>
				<div type="sentence">
					The amount of c-Rafin the immunoprecipitatenormalized to total input levelswas significantly higher with beta-arrestin2-Ub than with the wild type as indicatedby the quantification of bands from three independent experiments (data not shown).
					<desc type="ontological">protein(c-Raf)</desc>
					<desc type="ontological">method(immunoprecipitate)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					Previous studies have also shownthat by merely coexpressing beta-arrestin2with a MAPKKK (such as c-Raf, ASK -1) and a MAPK(such as ERK2, JNK3), robust activation of MAPKcould be achieved (18,24,25).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(c-Raf)</desc>
					<desc type="ontological">protein(ASK -1)</desc>
					<desc type="ontological">proteinOrGene(MAPK)</desc>
					<desc type="ontological">protein(ERK2)</desc>
					<desc type="ontological">protein(JNK3)</desc>
					<desc type="ontological">proteinOrGene(MAPK)</desc>
					<desc type="reference">reference(18)</desc>
					<desc type="reference">reference(24)</desc>
					<desc type="reference">reference(25)</desc>
				</div>
				<div type="sentence">
					This property of beta-arrestin2is attributed to its capacity to simultaneously bind component enzymes of a kinase cascade thus bringing them in to proximity and allowing robust phosphorylation to occur.
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					Accordingly, cotransfectionof beta-arrestin2, cRafand GFP-ERK2could enhance precipitation of phosphorylated ERK2with FLAG-beta-arrestin2(18).
					<desc type="ontological">method(cotransfection)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(cRaf)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="ontological">protein(ERK2)</desc>
					<desc type="relation">complex(GFP-ERK2)</desc>
					<desc type="relation">complexElement(GFP, GFP-ERK2)</desc>
					<desc type="relation">complexElement(ERK2, GFP-ERK2)</desc>
					<desc type="ontological">protein(ERK2)</desc>
					<desc type="relation">phosphoForm(p_ERK2, ERK2, unknownPosition)</desc>
					<desc type="action">reactionModulationInit(GFP-ERK2, p_ERK2, ERK2, unknownPosition, increase, unknownDistance, hypothesis, unknownCell, unknownMethod)</desc>
					<desc type="ontological">gene(FLAG)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="relation">complex(FLAG-beta-arrestin2)</desc>
					<desc type="relation">complexElement(FLAG, FLAG-beta-arrestin2)</desc>
					<desc type="relation">complexElement(beta-arrestin2, FLAG-beta-arrestin2)</desc>
					<desc type="reference">reference(18)</desc>
				</div>
				<div type="sentence">
					To examine whether beta-arrestin2 ^0Kwas capable of a similar function, we transfectedCOS-7cells with either WT, beta-arrestin2 ^0Kor beta-arrestin2-Ub along with RFP-ERK2and increasingamounts of myc-cRaf -1.
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">method(transfected)</desc>
					<desc type="ontological">cell(COS-7)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(RFP)</desc>
					<desc type="ontological">protein(ERK2)</desc>
					<desc type="relation">complex(RFP-ERK2)</desc>
					<desc type="relation">complexElement(RFP, RFP-ERK2)</desc>
					<desc type="relation">complexElement(ERK2, RFP-ERK2)</desc>
					<desc type="ontological">peptide(myc)</desc>
					<desc type="ontological">gene(cRaf -1)</desc>
					<desc type="relation">complex(myc-cRaf -1)</desc>
					<desc type="relation">complexElement(myc, myc-cRaf -1)</desc>
					<desc type="relation">complexElement(cRaf -1, myc-cRaf -1)</desc>
					<desc type="action">associationModulationInit(beta-arrestin2, RFP-ERK2, myc-cRaf -1, unknownparticle, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					As shownin the Western blots(Fig 9B) and the bargraphsdepicting quantification of pERKin beta-arrestin2precipitates (Fig 9C), beta-arrestin2 ^0Kcould scaffold pERKto the same extent as the WT.
					<desc type="ontological">method(Western blots)</desc>
					<desc type="reference">referenceFigure(Fig 9B)</desc>
					<desc type="ontological">gene(bar)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="reference">referenceFigure(Fig 9C)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">method(WT)</desc>
				</div>
				<div type="sentence">
					Interestingly, beta-arrestin2-Ub precipitates contained 60-80% more pERKthan the WTsuggesting either a greater levelof kinase activation and/or a stronger interaction of beta-arrestin2-Ub with pERK.
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(pERK)</desc>
				</div>
				<div type="sentence">
					All the above coimmunoprecipitationdata suggest that ubiquitination is not required for beta-arrestin'sinteraction with c-Rafand pERKand that despite the 31 lysinemutations beta-arrestin2 ^0Kcan interact with these beta-arrestin partners.
					<desc type="ontological">method(coimmunoprecipitation)</desc>
					<desc type="ontological">protein(c-Raf)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">molecule(lysine)</desc>
					<desc type="ontological">molecule(lysine)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					We next examined the effects of beta-arrestin2, beta-arrestin2-Ub, and beta-arrestin2 ^0Kon the assembly of receptor/beta-arrestin2/ERKcomplexes.
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(ERK)</desc>
					<desc type="relation">complex(beta-arrestin2-ERK)</desc>
					<desc type="relation">complexElement(beta-arrestin2, beta-arrestin2-ERK)</desc>
					<desc type="relation">complexElement(ERK, beta-arrestin2-ERK)</desc>
				</div>
				<div type="sentence">
					As depicted in Fig 10A, a significant amount of pERKwas associated with beta2ARimmunoprecipitatesfrom COS-7cells upon coexpression of beta-arrestin2-Ub.
					<desc type="reference">referenceFigure(Fig 10A)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">method(immunoprecipitates)</desc>
					<desc type="ontological">cell(COS-7)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					Lesser amounts of pERKwere detectedupon wild type beta-arrestin2expression, although this amount was still higher than the pERKdetectedwith endogenous beta-arrestin2in the mock-transfectedsamples (Fig 10, A and B).
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(beta-arrestin2)</desc>
					<desc type="ontological">process(expression)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">method(transfected)</desc>
					<desc type="reference">referenceFigure(Fig 10, A and B)</desc>
				</div>
				<div type="sentence">
					Expressionof beta-arrestin2 ^0Kresultedin a significant decreasein pERKin receptor complexes than what was obtainedwith endogenous beta-arrestin2as seen in the bar graphrepresenting the quantification of signals from five independent experiments (Fig 10B).
					<desc type="ontological">process(Expression)</desc>
					<desc type="ontological">gene(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="reference">referenceFigure(Fig 10B)</desc>
				</div>
				<div type="sentence">
					This decreasewas not due to a decline in overall ERKactivation since the levelof activation in whole celllysates was identical in all transfectionconditions.
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">compartment(whole cell)</desc>
					<desc type="ontological">method(transfection)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					In general, beta-arrestin-mediatedERKsignals are retained in the cytosoland are preventedfrom entering the nucleus.
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">compartment(cytosol)</desc>
					<desc type="action">localizationInit(ERK, cytosol, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">compartment(nucleus)</desc>
				</div>
				<div type="sentence">
					To determine if beta-arrestin ubiquitination plays a role in the subcellular localization of agonist-stimulatedpERK, we performed confocalimmunofluorescence microscopyand examined the relative distribution of agonist-activatedreceptors, beta-arrestins and pERK.
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">method(confocal)</desc>
					<desc type="ontological">method(immunofluorescence microscopy)</desc>
					<desc type="ontological">gene(pERK)</desc>
				</div>
				<div type="sentence">
					An antibody that specifically recognizes Thr 202/Tyr204-phosphorylated ERK1 /2was employed to detectactivatedendogenous ERK.
					<desc type="ontological">molecule(Thr 202)</desc>
					<desc type="ontological">molecule(Tyr)</desc>
					<desc type="relation">complex(Thr 202-Tyr)</desc>
					<desc type="relation">complexElement(Thr 202, Thr 202-Tyr)</desc>
					<desc type="relation">complexElement(Tyr, Thr 202-Tyr)</desc>
					<desc type="ontological">protein(ERK1 /2)</desc>
					<desc type="relation">phosphoForm(p_ERK1 /2, ERK1 /2, unknownPosition)</desc>
					<desc type="ontological">protein(ERK)</desc>
				</div>
				<div type="sentence">
					If ubiquitination of beta-arrestin2indeed plays a role in determining the spatialdistribution of active ERK, then differences should be observedin the cellular distribution of pERKstimulatedin the presence of beta-arrestin2-Ub versus beta-arrestin2 ^0K.
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(spatial)</desc>
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="action">associationModulationInit(spatial, ERK, pERK, unknownparticle, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					As depicted in Fig 11A, unstimulated cells showa uniform cytosolicdistribution of beta-arrestin2-GFP(green), a membranedistribution of HA-beta2AR(blue) and a negligible amount of pERK(red).
					<desc type="reference">referenceFigure(Fig 11A)</desc>
					<desc type="ontological">protein(cytosolic)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2, beta-arrestin2-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2-GFP)</desc>
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="action">transportModulationInit(cytosolic, beta-arrestin2-GFP, unknownlocalization, membrane, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">proteinOrGene(HA)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="relation">complex(HA-beta2)</desc>
					<desc type="relation">complexElement(HA, HA-beta2)</desc>
					<desc type="relation">complexElement(beta2, HA-beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="ontological">gene(pERK)</desc>
				</div>
				<div type="sentence">
					When the cells were stimulatedfor 5 minuteswith isoproterenol, beta-arrestin2redistributed to the cell membraneto colocalize with the activatedreceptors.
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">compartment(cell membrane)</desc>
					<desc type="action">localizationInit(beta-arrestin2, cell membrane, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					A robust increasein the levelof pERKwas observedin both cytosoland nucleusalong with a clearly demarcated pERKsignal on beta-arrestin-studded cell membranes(2^nd row, Fig 11A).
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">compartment(cytosol)</desc>
					<desc type="ontological">compartment(nucleus)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">compartment(cell membranes)</desc>
					<desc type="reference">referenceFigure(Fig 11A)</desc>
				</div>
				<div type="sentence">
					After 30 minutesof isoproterenol, the beta2ARswere visualized in intracellularvesicles.
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">gene(ARs)</desc>
					<desc type="relation">complex(beta2-ARs)</desc>
					<desc type="relation">complexElement(beta2, beta2-ARs)</desc>
					<desc type="relation">complexElement(ARs, beta2-ARs)</desc>
					<desc type="ontological">compartment(intracellular)</desc>
					<desc type="action">transportModulationInit(isoproterenol, beta2-ARs, unknownlocalization, intracellular, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					beta-arrestins are not localized to these vesicular structures but are retained at the cell membrane.
					<desc type="ontological">compartment(cell membrane)</desc>
				</div>
				<div type="sentence">
					A small percentage of receptors persist at the membrane, which most likely represent recycled and/or non-internalizedreceptors.
					<desc type="ontological">compartment(membrane)</desc>
				</div>
				<div type="sentence">
					After 30 minutesof isoproterenoltreatment, a negligible amount of pERKwas detected(3^rdrow, Fig 11A).
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">method(treatment)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">gene(rd)</desc>
					<desc type="reference">referenceFigure(Fig 11A)</desc>
				</div>
				<div type="sentence">
					As a comparison, representative cells overexpressingboth HA-beta2ARand beta-arrestin2-GFPtreatedwith phorbol myristate acetate (PMA) are shownin the bottom row of Fig 11A.
					<desc type="ontological">proteinOrGene(HA)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="relation">complex(HA-beta2)</desc>
					<desc type="relation">complexElement(HA, HA-beta2)</desc>
					<desc type="relation">complexElement(beta2, HA-beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2, beta-arrestin2-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2-GFP)</desc>
					<desc type="ontological">method(treated)</desc>
					<desc type="reference">referenceFigure(Fig 11A)</desc>
				</div>
				<div type="sentence">
					PMA stimulation leads to robust activation of ERK, which is distributed in both cytoplasmand nucleus.
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">compartment(cytoplasm)</desc>
					<desc type="ontological">compartment(nucleus)</desc>
					<desc type="action">localizationInit(ERK, cytoplasm, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="action">localizationInit(ERK, nucleus, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					PMA stimulation does not lead to either beta2ARinternalization or beta-arrestin2translocation.
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					The resultsof similar experiments performed with GFP-beta-arrestin2-Ub and HA-beta2ARsare shownin Fig 11B.
					<desc type="ontological">protein(GFP)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="relation">complex(GFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(GFP, GFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(beta-arrestin2, GFP-beta-arrestin2)</desc>
					<desc type="ontological">proteinOrGene(HA)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="relation">complex(HA-beta2)</desc>
					<desc type="relation">complexElement(HA, HA-beta2)</desc>
					<desc type="relation">complexElement(beta2, HA-beta2)</desc>
					<desc type="ontological">gene(ARs)</desc>
					<desc type="reference">referenceFigure(Fig 11B)</desc>
				</div>
				<div type="sentence">
					Under unstimulated conditions the sub-cellular distributions are identical to what is observedwith the WTbeta-arrestin2.
					<desc type="ontological">gene(sub)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					Quite strikingly, at 5-minutestimulation, a distinct and robust ERKactivation is observedat the cell membranecoinciding with the distinct membranerecruitmentof beta-arrestin2-Ub.
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">compartment(cell membrane)</desc>
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="ontological">process(recruitment)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					Although a majority of the cells (~80%) displayed such distribution at the cell membrane, some cells did have small vesicles in the vicinity of the cell membrane, which contained beta-arrestin2-Ub, beta2ARas well as pERKas shownin the figure panels (2^nd row, Fig 11B).
					<desc type="ontological">compartment(cell membrane)</desc>
					<desc type="ontological">compartment(cell membrane)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="reference">referenceFigure(Fig 11B)</desc>
				</div>
				<div type="sentence">
					Surprisingly at the 5 minutetime point, unlike the caseof WTbeta-arrestin2expression, little active ERKwas distributed in the nucleuswith beta-arrestin2-Ub overexpression.
					<desc type="ontological">gene(case)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(beta-arrestin2)</desc>
					<desc type="ontological">process(expression)</desc>
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">compartment(nucleus)</desc>
					<desc type="action">localizationInit(ERK, nucleus, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					We do not know the exact mechanism by which this occurs, but possibly, beta-arrestin2-Ub can simultaneously promotebeta-arrestin-dependent cytosolicERKand curbthe Gprotein ERKpathway leading to lesser nuclearERK.
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(cytosolic)</desc>
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">gene(curb)</desc>
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">compartment(nuclear)</desc>
					<desc type="action">transportModulationPerturbatorInit(ERK, curb, ERK, unknownLocalization, nuclear, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">protein(ERK)</desc>
				</div>
				<div type="sentence">
					After 30 minutesof isoproterenoltreatment, a dramatic redistribution of beta-arrestin2-Ub, beta2ARand pERKwas seen in intracellularvesicles.
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">method(treatment)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">compartment(intracellular)</desc>
					<desc type="action">transportModulationInit(beta2-AR, pERK, unknownlocalization, intracellular, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					These data clearly indicatethat a stably ubiquitinatedbeta-arrestin can remain associated with a 'Class A'receptor(i.e.
					<desc type="ontological">gene(A')</desc>
					<desc type="ontological">protein(A' receptor)</desc>
				</div>
				<div type="sentence">
					beta2AR) and target activatedERKto early endosomesresultingin a pool of pERKcomplexed with internalized receptors
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">protein(ERK)</desc>
					<desc type="action">quantityModulationInit(beta2-AR, ERK, increase, unknownDistance, confirmed)</desc>
					<desc type="ontological">compartment(early endosomes)</desc>
					<desc type="ontological">gene(pERK)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					In the absence of agonist, beta-arrestin2 ^0K-GFPis mainly cytoplasmic, with HA-beta2ARat the plasma membraneand very little active ERK(toprow, Fig 11C).
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(GFP)</desc>
					<desc type="relation">complex(beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(beta-arrestin2 ^0K, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="relation">complexElement(GFP, beta-arrestin2 ^0K-GFP)</desc>
					<desc type="ontological">compartment(cytoplasmic)</desc>
					<desc type="action">localizationInit(beta-arrestin2 ^0K-GFP, cytoplasmic, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">proteinOrGene(HA)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="relation">complex(HA-beta2)</desc>
					<desc type="relation">complexElement(HA, HA-beta2)</desc>
					<desc type="relation">complexElement(beta2, HA-beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="ontological">compartment(plasma membrane)</desc>
					<desc type="action">localizationInit(AR, plasma membrane, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">gene(top)</desc>
					<desc type="reference">referenceFigure(Fig 11C)</desc>
				</div>
				<div type="sentence">
					After 5 minutesof isoproterenol-stimulation, a robust activation of ERKoccurs which is seen distributed in both cytoplasmicand nuclearcompartments.
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">compartment(cytoplasmic)</desc>
					<desc type="ontological">compartment(nuclear)</desc>
					<desc type="action">localizationInit(ERK, cytoplasmic, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="action">localizationInit(ERK, nuclear, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					However, none of this active ERKis localized with beta-arrestin2 ^0K.
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
				</div>
				<div type="sentence">
					Possibly, much of this activity is Gprotein mediatedand is excluded from receptor complexes since less pERKis complexed with the beta2ARin the presence of beta-arrestin2 ^0K(see Fig 10).
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="reference">referenceFigure(Fig 10)</desc>
				</div>
				<div type="sentence">
					At 30 minutes, levelsof pERKdecreasedbut were not abolished(bottom row, Fig 11C).
					<desc type="ontological">gene(pERK)</desc>
					<desc type="reference">referenceFigure(Fig 11C)</desc>
				</div>
				<div type="sentence">
					This situation contrasts with what is observedwith the stably ubiquitinatedbeta-arrestin2-Ub (Fig 11B), where pERKsignals are stabilized and localized on endosomalvesicles at 30 minof isoproterenolstimulation.
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="reference">referenceFigure(Fig 11B)</desc>
					<desc type="ontological">gene(pERK)</desc>
					<desc type="ontological">compartment(endosomal)</desc>
					<desc type="action">transportModulationInit(beta-arrestin2, pERK, unknownlocalization, endosomal, increase, unknownDistance, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="relation">modifiedForm(isoproterenol_inactive, isoproterenol_active)</desc>
				</div>
				<div type="sentence">
					As seen in the 30 minpanels of Fig 11C, beta2ARinternalized into endosomeswhich is consistent with our internalization data (Fig 7AD), which indicatethe inability of beta-arrestin2 ^0Kto inhibitreceptor internalization.
					<desc type="reference">referenceFigure(Fig 11C)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">compartment(endosomes)</desc>
					<desc type="action">localizationInit(beta2-AR, endosomes, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="reference">referenceFigure(Fig 7)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					We also determined the kinetics of ERKphosphorylation in HEK-293cells expressingthe beta2AR(1 pmolper mgcellular protein) upon transfectionof vector, beta-arrestin2WT, beta-arrestin2 ^0Kor beta-arrestin2-Ub.
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">cell(HEK-293)</desc>
					<desc type="ontological">gene(beta2)</desc>
					<desc type="ontological">protein(beta2)</desc>
					<desc type="ontological">protein(AR)</desc>
					<desc type="relation">complex(beta2-AR)</desc>
					<desc type="relation">complexElement(beta2, beta2-AR)</desc>
					<desc type="relation">complexElement(AR, beta2-AR)</desc>
					<desc type="ontological">method(transfection)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">method(WT)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
				</div>
				<div type="sentence">
					As shownin Fig 12, expressionof beta-arrestin2-Ub significantly increasedERKactivity at 20 minof isoproterenoltreatment, beta-arrestin2led to a modest augmentation and beta-arrestin2 ^0Khad no effect over mock conditions (Fig 12B).
					<desc type="reference">referenceFigure(Fig 12)</desc>
					<desc type="ontological">process(expression)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(beta-arrestin2)</desc>
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">molecule(isoproterenol)</desc>
					<desc type="ontological">method(treatment)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2 ^0K)</desc>
					<desc type="reference">referenceFigure(Fig 12B)</desc>
				</div>
				<div type="sentence">
					Previous studies have demonstratedthat, later ERKactivity inducedby 7TMRsis actually beta-arrestin-mediated(reviewed in (26)).
					<desc type="ontological">protein(ERK)</desc>
					<desc type="ontological">protein(TMRs)</desc>
					<desc type="action">quantityModulationInit(ERK, TMRs, increase, unknownDistance, biblio)</desc>
					<desc type="reference">reference(26)</desc>
				</div>
				<div type="sentence">
					These resultsfurther support the idea that beta-arrestin ubiquitination status underlies some aspects of beta-arrestin-dependent signaling.
				</div>
			</div>
			<div type="paragraph">
				<div type="sentence">
					The beta-arrestin isoforms are mainly cytosolicproteins and are translocated to the plasma membraneupon 7TMRactivation.
					<desc type="ontological">protein(cytosolic)</desc>
					<desc type="ontological">compartment(plasma membrane)</desc>
					<desc type="action">localizationInit(cytosolic, plasma membrane, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">protein(TMR)</desc>
				</div>
				<div type="sentence">
					Thus farno lipid modifications in beta-arrestins favoring macromolecular membraneinteractions have been identified.
					<desc type="ontological">gene(far)</desc>
					<desc type="ontological">compartment(membrane)</desc>
				</div>
				<div type="sentence">
					One well accepted mechanism that keeps them in a membraneenvironment is their binding to phosphorylateddomains of receptors (5).
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="reference">reference(5)</desc>
				</div>
				<div type="sentence">
					Our current and previous resultsindicatethat ubiquitination could be an important factor that determines the longevity of beta-arrestin'sinteractions with receptorsleading to colocalizationon endosomalvesicles.
					<desc type="ontological">method(colocalization)</desc>
					<desc type="ontological">compartment(endosomal)</desc>
				</div>
				<div type="sentence">
					Interestingly, when we analyzed the distribution of the ubiquitinatedform of beta-arrestin by sub-cellular fractionation, we foundthat the ubiquitination status of beta-arrestin favors its partitioning to membranefractions.
					<desc type="ontological">gene(sub)</desc>
					<desc type="ontological">method(fractionation)</desc>
					<desc type="ontological">compartment(membrane)</desc>
				</div>
				<div type="sentence">
					When COS-7cells expressingeither beta-arrestin2or beta-arrestin2-Ub were lysed in a detergent free low saltbuffer (40 mMNaCl) and the soluble and insoluble fractions were further separated by differential centrifugation, nonubiquitinated beta-arrestins were mainly cytosolic.
					<desc type="ontological">cell(COS-7)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">gene(salt)</desc>
					<desc type="ontological">method(centrifugation)</desc>
					<desc type="ontological">protein(cytosolic)</desc>
				</div>
				<div type="sentence">
					Most of the exogenously expressedbeta-arrestin2as well as YFP-beta-arrestin2were detectable in the soluble fraction (Fig 13).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">protein(YFP)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="relation">complex(YFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(YFP, YFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(beta-arrestin2, YFP-beta-arrestin2)</desc>
					<desc type="reference">referenceFigure(Fig 13)</desc>
				</div>
				<div type="sentence">
					The YFP-beta-arrestin2band in the membranefraction with a slightly slower mobility is unreactive to ubiquitin antibodies such as FK 2, P4D1and FK1and its identity remains to be elucidated.
					<desc type="ontological">protein(YFP)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="relation">complex(YFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(YFP, YFP-beta-arrestin2)</desc>
					<desc type="relation">complexElement(beta-arrestin2, YFP-beta-arrestin2)</desc>
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="action">localizationInit(YFP-beta-arrestin2, membrane, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="ontological">gene(FK 2)</desc>
					<desc type="ontological">protein(P4)</desc>
					<desc type="ontological">protein(D1)</desc>
					<desc type="relation">complex(P4-D1)</desc>
					<desc type="relation">complexElement(P4, P4-D1)</desc>
					<desc type="relation">complexElement(D1, P4-D1)</desc>
					<desc type="ontological">gene(FK1)</desc>
				</div>
				<div type="sentence">
					On the other hand, ubiquitinatedbeta-arrestin2was distributed mostly in the insoluble membranefractions (Fig 13 A and B).
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="action">localizationInit(beta-arrestin2, membrane, confirmed, unknownCell, unknownMethod)</desc>
					<desc type="reference">referenceFigure(Fig 13 A and B)</desc>
				</div>
				<div type="sentence">
					As seen in Fig 4B, beta-arrestin2-Ub appears to be uniformly distributed in the cytosolin an undisturbed cell.
					<desc type="reference">referenceFigure(Fig 4B)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">compartment(cytosol)</desc>
					<desc type="action">localizationInit(beta-arrestin2, cytosol, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					Accordingly, the membranefractionationof beta-arrestin2-Ub is not due to its presence in inclusion bodies, but rather due to its affinity for membranecomponents.
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="ontological">method(fractionation)</desc>
					<desc type="ontological">protein(beta-arrestin2)</desc>
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="action">localizationInit(beta-arrestin2, membrane, confirmed, unknownCell, unknownMethod)</desc>
				</div>
				<div type="sentence">
					These resultssuggest that ubiquitination increasesbeta-arrestin'spropensity for membraneassociation thus favoring beta-arrestin'sprolonged localization in membranemicrodomains.
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="ontological">compartment(membrane)</desc>
					<desc type="ontological">compartment(microdomains)</desc>
				</div>
				<div type="sentence">
					Although ubiquitination is dispensable for beta-arrestin'sinteractions with cytososlic partners, it may be necessary to facilitate the formation of functional 7TMR -beta-arrestin endocyticand signaling complexes in a membraneenvironment.
					<desc type="ontological">protein(TMR -beta)</desc>
					<desc type="ontological">process(endocytic)</desc>
					<desc type="ontological">compartment(membrane)</desc>
				</div>
			</div>