Internalization of the Hm1 muscarinic cholinergic receptor involves the third cytoplasmic loop

The m1 muscarinic receptor was previously shown to stimulate phosphatidyl inositol (PI) turnover and to internalize rapidly upon agonist activation. Three receptor mutants with large deletions of the third cytoplasmic loop (i3) of human Hm1, leaving only 11 and 8 amino acids at the amino and carboxy terminal junctions of i3, respectively, retained full ability to stimulate PI turnover, when expressed in U293 cells, but receptor internalization was greatly reduced in two mutants with deletions reaching close to the NH2 terminal of i3. We propose that a receptor domain located toward the amino terminal junction of i3 plays a role in Hm1 internalization.     

Internalization and down-regulation of muscarinic acetylcholine receptors in cerebellar granule cells of tenascin-gene deficient mice

The expression of tenascin-C on oligodendrocytes parallels the migration of granule cells in the developing cerebellum, indicating a role for tenascin-C as a guide for granule neurons to find their proper locations. In this study, cultured cerebellar granule neurons from tenascin-C-knockout mice were used to examine the role of tenascin-C in agonist-induced muscarinic acetylcholine receptor down-regulation. Exposure of granule cells from wild-type or tenascin-C-negative mice to the muscarinic acetylcholine receptor agonist carbachol (1 mM) resulted in normal sequestration of cell-surface muscarinic acetylcholine receptors as assessed by [3H]N-methylscopolamine binding; however, down-regulation of total muscarinic acetylcholine receptors, measured with [3H]quinuclidinyl benzilate, was inhibited in granule cells from tenascin-C-negative mice. Remarkably, incubation of the tenascin-C-negative cells with the microtubule stabilizer taxol (10 microM) restored down-regulation of total muscarinic acetylcholine receptors to normal levels. We speculate that agonist-induced down-regulation of muscarinic acetylcholine receptors is functionally associated with tenascin-C-regulated microtubule structures in the developing cerebellum.     

beta-arrestins regulate interleukin-8-induced CXCR1 internalization.

The functional role of neutrophils during acute inflammatory responses is regulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) that are rapidly desensitized and internalized upon binding their cognate chemokine ligands. The efficient re-expression of CXCR1 on the surface of neutrophils following agonist-induced internalization suggests that CXCR1 surface receptor turnover may involve regulatory pathways and intracellular factors similar to those regulating beta2-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligand-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two different cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimulation promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalization in HEK 293 cells required co-expression of G protein-coupled receptor kinase 2 and beta-arrestin proteins. The importance of beta-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative beta-arrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalization in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Agonist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin-coated vesicles. Our data provides a direct link between agonist-induced internalization of CXCR1 and a requirement for G protein-coupled receptor kinase 2, beta-arrestins, and dynamin during this process.     

Mutational analysis of third cytoplasmic loop domains in G-protein coupling of the HM1 muscarinic receptor.

We measured dose-response curves for carbachol stimulation of phosphatidyl inositol (PI) turnover with mutants of the Hm1 muscarinic cholinergic receptor having various deletions from amino acids 219 to 358 of the large third intracellular (i3) loop (208 to 366). These deletions had only small or no effects on the ability of Hm1 transfected into HEK 293 cells to stimulate PI turnover. This result indicates that only small regions of 9 to 11 amino acids adjacent to trans-membrane domains (TMDs) 5 and 6 can be directly involved in G protein coupling. Point mutations were constructed to test the role of charged amino acids in these junctions. A triple point mutation of Hm1 (E214 A/ E216K/ E221 K), which mimics the charge distribution in Hm2 (negatively coupled to cAMP) over the first 14 amino acids of i3, and a double point mutation in the N terminal junction, K359A/K361A, both failed to affect carbachol stimulated PI turnover. Therefore, charge distribution in the loop junctions appears to play a minor role in G protein coupling of Hm1 in HEK 293 cells.     

The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation.

The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules.     

Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.     

Identification of a motif in the carboxyl terminus of CXCR2 that is involved in adaptin 2 binding and receptor internalization

Agonist treatment of cells expressing the chemokine receptor, CXCR2, induces receptor phosphorylation and internalization through a dynamin-dependent mechanism. In the present study, we demonstrate that a carboxyl terminus-truncated mutant of CXCR2 (331T), which no longer undergoes agonist-induced phosphorylation, continues to undergo ligand-induced internalization in HEK293 cells. This mutant receptor exhibits reduced association with beta-arrestin 1 but continues to exhibit association with adaptin 2 alpha and beta subunits. Replacing Leu320-321 and/or Ile323-Leu324 with Ala (LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA) in wild-type CXCR2 or 331T causes little change in ligand binding and signaling through Ca(2+) mobilization but greatly impairs the agonist-induced receptor sequestration and ligand-mediated chemotaxis. The LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA mutants of CXCR2 exhibit normal binding to beta-arrestin 1 but exhibit decreased binding to adaptin 2alpha and beta. These data demonstrate a role for the LLKIL motif in the carboxyl terminus of CXCR2 in receptor internalization and cell chemotaxis and imply a role for adaptin 2 in the endocytosis of CXCR2.     

Beta-arrestin1 and beta-arrestin2 are differentially required for phosphorylation-dependent and -independent internalization of delta-opioid receptors

Beta-arrestins are key negative regulators and scaffolds of G protein-coupled receptor (GPCR) signalling. Beta-arrestin1 and beta-arrestin2 preferentially bind to the phosphorylated GPCRs in response to agonist stimulation, resulting in receptor internalization and desensitization. The critical roles of GPCR kinases (GRKs)-catalyzed receptor phosphorylation and interaction of beta-arrestins with the phosphorylated receptor in receptor internalization are well established. However, emerging evidence suggests that an agonist-stimulated internalization mechanism that is independent of receptor phosphorylation may also be employed in some cases, although the molecular mechanism for the phosphorylation-independent GPCR internalization is not clear. The current study investigated the role of receptor phosphorylation and the involvement of different beta-arrestin subtypes in agonist-induced delta-opioid receptor (DOR) internalization in HEK293 cells. Results from flow cytometry, fluorescence microscopy, and surface biotin labelling experiments showed that elimination of agonist-induced DOR phosphorylation by mutation GRK binding or phosphorylation sites only partially blocked agonist-induced receptor internalization, indicating the presence of an agonist-induced, GRK-independent mechanism for DOR internalization. Fluorescence and co-immunoprecipitation studies indicated that both the wild-type DOR and the phosphorylation-deficient mutant receptor could bind and recruit beta-arrestin1 and beta-arrestin2 to the plasma membrane in an agonist-stimulated manner. Furthermore, internalization of both the wild-type and phosphorylation-deficient receptors was increased by overexpression of either type of beta-arrestins and blocked by dominant-negative mutants of beta-arrestin-mediated internalization, demonstrating that both phosphorylation-dependent and -independent internalization require beta-arrestin. Moreover, double-stranded RNA-mediated interference experiments showed that either beta-arrestin1 or beta-arrestin2 subtype-specific RNAi only partially inhibited agonist-induced internalization of the wild-type DOR. However, agonist-induced internalization of the phosphorylation-deficient DOR was not affected by beta-arrestin1-specific RNAi but was blocked by RNAi against beta-arrestin2 subtype. These data indicate that endogenous beta-arrestin1 functions exclusively in the phosphorylation-dependent receptor internalization, whereas endogenous beta-arrestin2, but not beta-arrestin1, is required for the phosphorylation-independent receptor internalization. These results thus provide the first evidence of different requirement for beta-arrestin isoforms in the agonist induced phosphorylation-dependent and -independent GPCR internalization.     

Chimeric AT1/AT2 receptors reveal functional similarities despite key amino acid dissimilarities in the domains mediating agonist-dependent activation

Chimeric AT1/AT2 angiotensin II (AngII) receptors in which the sixth and/or seventh transmembrane-spanning domains of the AT2 receptor were substituted into the AT1 receptor were used to investigate the activation mechanisms of the two receptor subtypes. Numerous reports have identified amino acid residues in the sixth and seventh transmembrane-spanning domains of the AT1 receptor involved in the intrareceptor activation mechanism following agonist binding. Many of these residues are not conserved in the AT2 receptor; the corresponding AT2 receptor residues are, in fact, disruptive of AngII-dependent activation when substituted into the AT1 receptor. Surprisingly, the chimeric AT1/AT2 receptors--which also lack these crucial AT1 residues--exhibited AngII-induced activation of phosphoinositide hydrolysis with efficacies and potencies similar to the wild-type AT1 receptor. Consistent with earlier reports, a AT1[Y292F] point mutant demonstrated greatly decreased agonist-induced activation of phosphoinositide hydrolysis. However, a AT1[Y292F/N295S] double-point mutant allowed for normal agonist-induced activation with a pharmacodynamic profile indistinguishable from the wild-type receptor. Despite amino acid dissimilarities, the same corresponding domains and even the same residue loci in both of the AngII receptor subtypes are equally able to mediate agonist-induced receptor activation. This suggests that these corresponding domains in the AT1 and the AT2 receptors are crucial to the activation mechanism, demonstrating greater structural flexibility than previously believed regarding AT1 receptor activation and supporting the possibility of a common activation mechanism for the two receptor subtypes.     

Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors

After short preincubations with N-[(3)H]methylscopolamine ([(3)H]NMS) or R(-)-[(3)H]quinuclidinyl benzilate ([(3)H]QNB), radioligand dissociation from muscarinic M(1) receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [(3)H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [(3)H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M(1) receptors. Covalently binding [(3)H]propylbenzilylcholine mustard detected substantially more binding sites than [(3)H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M(1) receptors may include Asp-99. Experimental data on [(3)H]NMS and [(3)H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.     

Palmitoylation of the human bradykinin B2 receptor influences ligand efficacy.

To investigate the palmitoylation of the human bradykinin B2 receptor, we have mutated individually or simultaneously into glycine two potential acylation sites (cysteines 324 and 329) located in the carboxyl terminus of the receptor and evaluated the effects of these mutations by transfection in COS-7, CHO-K1, and HEK 293T. The wild-type receptor and the single mutants, but not the double mutant, incorporated [3H]palmitate, indicating that the receptor carboxyl tail can be palmitoylated at both sites. The mutants did not differ from the wild-type receptor for the kinetics of [3H]bradykinin binding, the basal and bradykinin-stimulated coupling to phospholipases C and A2, and agonist-induced phosphorylation. The nonpalmitoylated receptor had a 30% reduced capacity to internalize [3H]bradykinin. This indicates that palmitoylation does not influence the basal activity of the receptor and its agonist-driven activation. However, the mutants triggered phospholipid metabolism and MAP kinase activation in response to B2 receptor antagonists. Pseudopeptide and nonpeptide compounds that behaved as antagonists on the wild-type receptor became agonists on the nonpalmitoylated receptor and produced phospholipases C and A2 responses of 25-50% as compared to that of bradykinin. These results suggest that palmitoylation is required for the stabilization of the receptor-ligand complex in an uncoupled conformation.     

Pharmacological evaluation of the long-term effects of xanomeline on the M(1) muscarinic acetylcholine receptor

Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M(1) and M(4) receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of xanomeline on the binding characteristics and function of the M(1) muscarinic receptor. Pretreatment of CHO cells that stably express the M(1) receptor for 1 hr with increasing concentrations of xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed xanomeline-induced inhibition of [(3)H]NMS binding from monophasic to biphasic. The high-affinity xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound xanomeline developed with a time course that parallels the return of receptor activation by prebound xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of xanomeline, but not its persistent binding at an allosteric site. Furthermore, the long-term effects of xanomeline on the receptor are mainly due to receptor down-regulation rather than internalization.     

Effect of pH on Muscarinic Acetylcholine Receptors from Rat Brainstem

The effect of hydrogen ion concentration on ligand binding to muscarinic acetylcholine receptors was studied in membranes isolated from rat brainstem. The binding of [3H]methylscopolamine was constant between pH 7 and 10. The affinity, but not the number, of [3H]methylscopolamine binding sites decreased below pH 7; at pH 4 little binding was detected. When brainstem membranes were incubated at various pH levels from 3 to 11 for 1 h and then returned to pH 8, [3H]methylscopolamine binding affinity was restored to control levels. Carbamylcholine binding affinity was also depressed in media of low pH. However, this decrease was permanent after a 1-h incubation at pH 4 (i.e. carbamylcholine affinity was not restored on raising the pH to 8). The capacity of a guanine nucleotide to affect carbamylcholine was also abolished by a 1-h incubation at pH 4, and was not restored by raising the pH. The guanine nucleotide-dependent regulatory protein may be irreversibly inactivated or dissociated from the receptor at low pH. The receptor's binding subunit, on the other hand, appears to be much less sensitive to hydrogen ion concentration.     

Agonist-induced internalization of the platelet-activating factor receptor is dependent on arrestins but independent of G-protein activation. Role of the C terminus and the (D/N)PXXY motif.

As with most G-protein-coupled receptors, repeated agonist stimulation of the platelet-activating factor receptor (PAFR) results in its desensitization, sequestration, and internalization. In this report, we show that agonist-induced PAFR internalization is independent of G-protein activation but is dependent on arrestins and involves the interaction of arrestins with a limited region of the PAFR C terminus. In cotransfected COS-7 cells, both arrestin-2 and arrestin-3 could be coimmunoprecipitated with PAFR, and agonist stimulation of PAFR induced the translocation of both arrestin-2 and arrestin-3. Furthermore, coexpression of arrestin-2 with PAFR potentiated receptor internalization, whereas agonist-induced PAFR internalization was inhibited by a dominant negative mutant of arrestin-2. The coexpression of a minigene encoding the C-terminal segment of the receptor abolished PAF-induced arrestin translocation and inhibited PAFR internalization. Using C terminus deletion mutants, we determined that the association of arrestin-2 with the receptor was dependent on the region between threonine 305 and valine 330 because arrestin-2 could be immunoprecipitated with the mutant PAFRstop330 but not PAFRstop305. Consistently, stop330 could mediate agonist-induced arrestin-2 translocation, whereas stop305 could not. Two other deletion mutants with slightly longer regions of the C terminus, PAFRstop311 and PAFRstop317, also failed to induce arrestin-2 translocation. Finally, the PAFR mutant Y293A, containing a single substitution in the putative internalization motif DPXXY in the seventh transmembrane domain (which we had shown to be able to internalize but not to couple to G-proteins) could efficiently induce arrestin translocation. Taken together, our results indicate that ligand-induced PAFR internalization is dependent on arrestins, that PAFR can associate with both arrestin-2 and -3, and that their translocation involves interaction with the region of residues 318-330 in the PAFR C terminus but is independent of G-protein activation.     

Muscarinic Receptor Stimulation Increases Inositol-Phospholipid Metabolism and Inhibits Cyclic AMP Accumulation in PC 12 Cells

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

The association of arrestin-3 with the human lutropin/choriogonadotropin receptor depends mostly on receptor activation rather than on receptor phosphorylation.

Although the involvement of the nonvisual arrestins in the agonist-induced internalization of the human lutropin receptor (hLHR) has been documented previously with the use of dominant-negative mutants, a physical association of the nonvisual arrestins with the hLHR in intact cells has not been established. In the studies presented herein, we used a cross-linking/coimmunoprecipitation/immunoblotting approach as well as confocal microscopy to document the association of the hLHR with the nonvisual arrestins in co-transfected 293 cells. We also used this approach to examine the relative importance of receptor activation and receptor phosphorylation in the formation of this complex. Using hLHR mutants that impair phosphorylation, activation, or both, we show that the formation of the hLHR-nonvisual arrestin complex depends mostly on the agonist-induced activation of the hLHR rather than on the phosphorylation of the hLHR. These results stand in contrast to those obtained with several other G protein-coupled receptors (i.e. the beta2-adrenergic receptor, the m2 muscarinic receptor, rhodopsin, and the type 1A angiotensin receptor) where arrestin binding depends mostly on receptor phosphorylation rather than on receptor activation. We have also examined the association of the nonvisual arrestins with naturally occurring gain-of-function mutations of the hLHR found in boys with Leydig cell hyperplasia or Leydig cell adenomas. Our results show that these mutants associate with the nonvisual arrestins in an agonist-independent fashion.     

Regulation of muscarinic acetylcholine receptor sequestration and function by beta-arrestin

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic beta-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the beta2-adrenergic receptor have demonstrated that beta-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of beta-arrestin in mAChR sequestration, we determined the effect of overexpressing beta-arrestin-1 and the dominant-negative inhibitor of beta-arrestin-mediated receptor sequestration, beta-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60-75% in cells overexpressing beta-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of beta-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrin-coated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50-70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a beta-arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.     

Agonist-induced internalization and mitogen-activated protein kinase activation of the human prostaglandin EP4 receptor

We examined the pathway of prostaglandin E(2) (PGE(2))-induced internalization of the prostaglandin EP4 receptor in HEK 293 cells. Co-expression of dominant negative beta-arrestin (319-418) or dynamin I (K44A) with the EP4 receptor reduced internalization. The activated receptor co-localized with GFP-arrestin 2 and GFP-arrestin 3, confirming the requirement for beta-arrestins in internalization. Inhibition of clathrin-coated vesicle-mediated internalization resulted in inhibition of sequestration, whereas inhibition of caveola-mediated internalization had no effect. PGE(2) stimulation of the EP4 receptor resulted in rapid mitogen-activated protein (MAP) kinase activation. Examination of an internalization-resistant mutant and co-expression of mutant accessory proteins with EP4 revealed that MAP kinase activation proceeds independently of internalization.     

Expression levels strongly affect ligand-induced sequestration of B2 bradykinin receptors in transfected cells

Transfection of cells with expression vectors is one of the most important tools used to assess the effects of receptor mutations on ligand-induced receptor sequestration. Most transfection methods give rise to transiently or stably transfected clones with a wide range of receptor expression levels that may also depend on the mutations made. It is, therefore, important to determine how the regulation of the receptors depends on their numbers per cell. In Chinese hamster ovary (CHO) and human embryonic kidney (HEK)-293 cells expressing high levels of B(2) kinin receptors, we observed poor sequestration indicated by <20% reduction in cell surface receptor number after 10 min of stimulation with 1 microM bradykinin (BK) compared with >70% in low-expressing cells. Whereas the rate of [(3)H]BK internalization (internalized [(3)H]BK in percentage of total bound [(3)H]BK) in low-expressing cells was independent of the ligand-concentration used, in high-expressing cells a strong rate decrease was observed with higher (>1 nM) concentrations. Lower ligand concentrations, however, led to internalization rates identical to those obtained in low-expressing cells. Transiently transfected HEK and COS-7 cells showed results similar to those of stably high-expressing cells. Our results demonstrate the difficulty in determining the internalization pattern of (mutated) B(2) kinin receptors, and possibly of G protein-coupled receptors in general, using a sequestration assay in high-expressing cells or transiently transfected cells with high numbers of receptors per transfected cell. However, the receptor (mutant)-specific internalization rate can be measured, provided that the ligand concentrations used are below a threshold at which the internalization rate is still independent of the ligand concentration.     

Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1. A role in receptor sequestration

Agonist-dependent desensitization of the beta-adrenergic receptor requires translocation and activation of the beta-adrenergic receptor kinase1 by liberated Gbetagamma subunits. Subsequent internalization of agonist-occupied receptors occurs as a result of the binding of beta-arrestin to the phosphorylated receptor followed by interaction with the AP2 adaptor and clathrin proteins. Receptor internalization is known to require D-3 phosphoinositides that are generated by the action of phosphoinositide 3-kinase. Phosphoinositide 3-kinases form a family of lipid kinases that couple signals via receptor tyrosine kinases and G-protein-coupled receptors. The molecular mechanism by which phosphoinositide 3-kinase acts to promote beta-adrenergic receptor internalization is not well understood. In the present investigation we demonstrate a novel finding that beta-adrenergic receptor kinase 1 and phosphoinositide 3-kinase form a cytosolic complex, which leads to beta-adrenergic receptor kinase 1-mediated translocation of phosphoinositide 3-kinase to the membrane in an agonist-dependent manner. Furthermore, agonist-induced translocation of phosphoinositide 3-kinase results in rapid interaction with the receptor, which is of functional importance, since inhibition of phosphoinositide 3-kinase activity attenuates beta-adrenergic receptor sequestration. Therefore, agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane is an important step in the process of receptor sequestration and links phosphoinositide 3-kinase to G-protein-coupled receptor activation and sequestration.