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J L Benovic

Publications and source records attributed to J L Benovic.

At least 37 records · Page 2Linked to original sources
PubMed2000

Selective regulation of endogenous G protein-coupled receptors by arrestins in HEK293 cells.

Arrestins play an important role in regulating desensitization and trafficking of G protein-coupled receptors (GPCRs). However, limited insight into the specificity of arrestin-mediated regulation of GPCRs is currently available. Recently, we used an antisense strategy to reduce arrestin levels in HEK293 cells and characterize the role of arrestins on endogenous G(s)-coupled receptors (Mundell, S. J., Loudon, R. B., and Benovic, J. L. (1999) Biochemistry 38, 8723-8732). Here, we characterized GPCRs coupled to either G(q) (M(1) muscarinic acetylcholine receptor (M(1)AchR) and P2y(1) and P2y(2) purinergic receptors) or G(i) (somatostatin and AT1 angiotensin receptors) in wild type and arrestin antisense HEK293 cells. The agonist-specific desensitization of the M(1)Ach and somatostatin receptors was significantly attenuated in antisense-expressing cells, whereas desensitization of P2y(1) and P2y(2) purinergic and AT1 angiotensin receptors was unaffected by reduced arrestin levels. To further examine arrestin/GPCR specificity, we studied the effects of endogenous GPCR activation on the redistribution of arrestin-2 epitope tagged with the green fluorescent protein (arrestin-2-GFP). These studies revealed a receptor-specific movement of arrestin-2-GFP that mirrored the arrestin-receptor specificity observed in the antisense cells. Thus, agonist-induced activation of endogenous beta(2)-adrenergic, prostaglandin E(2), M(1)Ach, and somatostatin receptors induced arrestin-2-GFP redistribution to early endosomes, whereas P2y(1) and P2y(2) purinergic and AT1 angiotensin receptor activation did not. Thus, endogenous arrestins mediate the regulation of selective G(q)- and G(i)-coupled receptors in HEK293 cells.

Adenosine Diphosphate↗
PubMed2000

Mutational analysis of Gbetagamma and phospholipid interaction with G protein-coupled receptor kinase 2.

Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free Gbetagamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with Gbetagamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by Gbetagamma. Alternatively, mutation of residues distal to the C-terminal amphipathic alpha-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to Gbetagamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with Gbetagamma, resulted in a specific and profound loss of Gbetagamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound Gbetagamma similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind Gbetagamma. In order to examine the role of phospholipid and Gbetagamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta(2)-adrenergic receptor (beta(2)AR) mutant that is responsive to GRK2 phosphorylation (beta(2)AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta(2)AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for Gbetagamma and phospholipids, and (ii) demonstrated that Gbetagamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.

Amino Acid Sequence↗
PubMed2000

Identification of Gbetagamma binding sites in the third intracellular loop of the M(3)-muscarinic receptor and their role in receptor regulation.

Gbetagamma binds directly to the third intracellular (i3) loop subdomain of the M(3)-muscarinic receptor (MR). In this report, we identified the Gbetagamma binding motif and G-protein-coupled receptor kinase (GRK2) phosphorylation sites in the M(3)-MR i3 loop via a strategy of deletional and site-directed mutagenesis. The Gbetagamma binding domain was localized to Cys(289)-His(330) within the M(3)-MR-Arg(252)-Gln(490) i3 loop, and the binding properties (affinity, influence of ionic strength) of the M(3)-MR-Cys(289)-His(330) i3 loop subdomain were similar to those observed for the entire i3 loop. Site-directed mutagenesis of the M(3)-MR-Cys(289)-His(330) i3 loop subdomain indicated that Phe(312), Phe(314), and a negatively charged region (Glu(324)-Asp(329)) were required for interaction with Gbetagamma. Generation of the full-length M(3)-MR-Arg(252)-Gln(490) i3 peptides containing the F312A mutation were also deficient in Gbetagamma binding and exhibited a reduced capacity for phosphorylation by GRK2. A similar, parallel strategy resulted in identification of major residues ((331)SSS(333) and (348)SASS(351)) phosphorylated by GRK2, which were just downstream of the Gbetagamma binding motif. Full-length M(3)-MR constructs lacking the 42-amino acid Gbetagamma binding domain (Cys(289)-His(330)) or containing the F312A mutation exhibited ligand recognition properties similar to wild type receptor and also effectively mediated agonist-induced increases in intracellular calcium following receptor expression in Chinese hamster ovary and/or COS 7 cells. However, the M(3)-MRDeltaCys(289)-His(330) and M(3)-MR(F312A) constructs were deficient in agonist-induced sequestration, indicating a key role for the Gbetagamma-M(3)-MR i3 loop interaction in receptor regulation and signal processing.

Amino Acid Sequence↗
PubMed2000

Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin.

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca(2+)-binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca(2+) coordination and the fact that their central alpha-helixes are extended by one alpha-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca(2+)-mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.

Amino Acid Sequence↗
PubMed2000

Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking.

G-protein-coupled receptors (GPCRs) mediate a diverse array of biological functions as a result of their ability to respond selectively to extracellular stimuli, which ultimately results in cell-specific activation of signaling cascades. Generally, GPCR activation is followed rapidly by a loss of responsiveness, termed desensitization, which is then followed by a period of recovery or resensitization. These changes in signaling potential are tightly regulated, primarily via mechanisms that involve GPCR phosphorylation and trafficking to distinct locations within the cell. Tagging of GPCRs with the green fluorescent protein (GFP) has enabled the direct visualization of real-time trafficking of GPCRs in living cells. Such analyses have provided crucial insight into the mechanisms involved in controlling GPCR function.

Animals↗
PubMed2000

Regulation of G protein-coupled receptor kinases.

G protein-coupled receptor kinases (GRKs) specifically interact with the agonist-activated form of G protein-coupled receptors (GPCRs) to effect receptor phosphorylation and desensitization. Recent studies demonstrate that GRK function is a highly regulated process, and it is perhaps in this manner that a handful of GRKs (7 have been identified to date) are able to regulate the responsiveness of numerous GPCRs in a given cell type in a coordinated manner. The mechanisms by which GRK activity is regulated can be divided into 3 categories: 1) subcellular localization; 2) alterations in intrinsic kinase activity; and 3) alterations in GRK expression levels. This review will summarize our current understanding of each of these regulatory processes, and offer explanations as to how such mechanisms influence GPCR regulation under various physiologic conditions.

Animals↗
PubMed2000

G-Protein-coupled receptor kinase activity in hypertension : increased vascular and lymphocyte G-protein receptor kinase-2 protein expression.

Impaired receptor-stimulated adenylyl cyclase activation has been observed in lymphocytes from hypertensive subjects and has been linked to an increase in lymphocyte G-protein receptor kinase-2 (GRK-2) protein expression. However, whether the increase in lymphocyte GRK-2 reflected an increase in vascular GRK-2 was unknown. Therefore, we compared GRK-2 protein expression in lymphocytes and aortas obtained from normotensive Wistar rats, Wistar-Kyoto rats (WKY), and spontaneously hypertensive rats (SHR) and from aortas of Dahl rats. Impaired beta-adrenergic responsiveness was observed in lymphocytes and vascular tissues obtained from hypertensive SHR (10 and 15 weeks old) but not in those obtained from prehypertensive SHR (5 weeks old). Immunodetectable lymphocyte GRK-2 protein expression was increased in 10-week-old SHR (143+/-10% of the expression in 10-week-old Wistar rats and 131+/-11% of the expression in 10-week-old WKY, n=5 in each group). Immunodetectable vascular smooth muscle cell GRK-2 was comparably increased (169+/-14% of the expression in Wistar rats and 138+/-7% of the expression in WKY, n=5 in each group). Also, in hypertensive Dahl salt-sensitive rats, vascular GRK-2 protein expression was increased (185+/-14% of the expression in Dahl salt-resistant rats, n=5 in each group) compared with Dahl salt-resistant controls. These studies support a generalized defect in vascular GRK-2 protein expression in hypertension, which could be an important factor in the impairment of beta-adrenergic-mediated vasodilation, characteristic of the hypertensive state.

Adenylyl Cyclases↗
PubMed2000

Mechanisms of proliferation synergy by receptor tyrosine kinase and G protein-coupled receptor activation in human airway smooth muscle.

Despite recent studies depicting the capacity of G protein-coupled receptors (GPCRs) to activate mitogenic signaling pathways more commonly associated with receptor tyrosine kinases (RTKs), little is known regarding the interactive effects of GPCR and RTK activation on cell growth and signal transduction. Such interactions likely mediate the physiologic growth in most cells in vivo as well as the aberrant, non-neoplastic growth that occurs in diseases such as asthma, where disruptions of the local hormonal or inflammatory state can contribute to significant GPCR activation. In this study, we show that numerous inflammatory or contractile agents, including thrombin, histamine, and carbachol, potentiate epidermal growth factor (EGF)-stimulated proliferation of human airway smooth muscle (ASM), thus demonstrating a clear synergy between RTK and GPCR activation. Alterations in promitogenic nuclear signaling were evidenced by additive or synergistic increases in Elk-1 and activator protein-1 activation, and by increases in cyclin D1 expression. Interestingly, GPCR activation did not cause EGF receptor tyrosine phosphorylation nor did it increase EGF-stimulated autophosphorylation. In the presence of EGF, histamine or carbachol did not alter the time-dependent phosphorylation of p42/p44, whereas thrombin was capable of increasing phospho-p42/p44 levels at selected time points in some, but not all, cultures. In contrast to their relative inability to alter EGF receptor-linked p42/p44 activation, thrombin, histamine, and carbachol consistently increased the late phase (> 1 h) activity of p70 S6 kinase. Collectively, these findings suggest that inflammatory and contractile agents that activate GPCRs can significantly modulate RTK-mediated ASM growth through a p70 S6 kinase-dependent, p42/p44-independent mechanism.

Carbachol↗
PubMed2000

Selective recruitment of arrestin-3 to clathrin coated pits upon stimulation of G protein-coupled receptors.

Non-visual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G protein-coupled receptors. In vitro experiments have shown that both non-visual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonist-promoted internalization of many receptors has been found to be stimulated by exogenous over-expression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When (beta)(2)-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G protein-coupled receptors to a specific non-visual arrestin in an in vivo setting.

Arrestins↗
PubMed2000

Mechanisms of agonist-induced down-regulation of the human kappa-opioid receptor: internalization is required for down-regulation.

Previously, we showed that the human kappa-opioid receptor (hkor) stably expressed in Chinese hamster ovary (CHO) cells underwent down-regulation after prolonged U50,488H treatment. In the present study, we determined the mechanisms underlying this process. U50, 488H caused a significant down-regulation of the hkor, although etorphine did not. Neither U50,488H nor etorphine caused down-regulation of the rat kappa-opioid receptor. Thus, similar to internalization, there are agonist and species differences in down-regulation of kappa-opioid receptors. Expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A significantly reduced U50,488H-induced down-regulation of the hkor. Coexpression of GRK2 or GRK2 and arrestin-2 permitted etorphine to induce down-regulation of the hkor, although expression of arrestin-2 or dynamin I alone did not. Expression of the dominant negative mutants rab5A-N133I or rab7-N125I blunted U50,488H-induced down-regulation. Pretreatment with lysosomal enzyme inhibitors [(2S, 3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or chloroquine] or proteasome inhibitors (proteasome inhibitor I, MG-132, or lactacystin) decreased the extent of U50,488H-induced down-regulation. A combination of chloroquine and proteasome inhibitor I abolished U50,488H-induced down-regulation. These results indicate that U50,488H-induced down-regulation of the hkor involves GRK-, arrestin-2-, dynamin-, rab5-, and rab7-dependent mechanisms and receptors seem to be trafficked to lysosomes and proteasomes for degradation. Thus, U50,488H-induced internalization and down-regulation of the hkor share initial common mechanisms. To the best of our knowledge, these results represent the first report on the involvement of both rab5 and rab7 in agonist-induced down-regulation of a G protein-coupled receptor. In addition, this study is among the first to show the involvement of proteasomes in agonist-induced down-regulation of a G protein-coupled receptor.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1999

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2.

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.

Aluminum Compounds↗
PubMed1999

Trafficking of the HIV coreceptor CXCR4. Role of arrestins and identification of residues in the c-terminal tail that mediate receptor internalization.

The G protein-coupled chemokine receptor CXCR4 serves as the primary coreceptor for entry of T-cell tropic human immunodeficiency virus. CXCR4 undergoes tonic internalization as well as internalization in response to stimulation with phorbol esters and ligand (SDF-1alpha). We investigated the trafficking of this receptor, and we attempted to define the residues of CXCR4 that were critical for receptor internalization. In both COS-1 and HEK-293 cells transiently overexpressing CXCR4, SDF-1alpha and phorbol esters (PMA) promoted rapid internalization of cell surface receptors as assessed by both enzyme-linked immunosorbent assay and immunofluorescence analysis. Expression of GRK2 and/or arrestins promoted modest additional CXCR4 internalization in response to both PMA and SDF. Both PMA- and SDF-mediated CXCR4 internalization was inhibited by coexpression of dominant negative mutants of dynamin-1 and arrestin-3. Arrestin was also recruited to the plasma membrane and appeared to colocalize with internalized receptors in response to SDF but not PMA. We then evaluated the ability of CXCR4 receptors containing mutations of serines and threonines, as well as a dileucine motif, within the C-terminal tail to be internalized and phosphorylated in response to either PMA or SDF-1alpha. This analysis showed that multiple residues within the CXCR4 C-terminal tail appear to mediate both PMA- and SDF-1alpha-mediated receptor internalization. The ability of coexpressed GRK2 and arrestins to promote internalization of the CXCR4 mutants revealed distinct differences between respective mutants and suggested that the integrity of the dileucine motif (Ile-328 and Leu-329) and serines 324, 325, 338, and 339 are critical for receptor internalization.

Amino Acid Sequence↗
PubMed1999

Characterization of G protein-coupled receptor regulation in antisense mRNA-expressing cells with reduced arrestin levels.

Previous studies with overexpressing wild-type or dominant negative nonvisual arrestins have established a role for these proteins in beta2-adrenergic receptor (beta2AR) internalization, desensitization, and resensitization. To validate and extend such findings, we employed an antisense strategy to target the nonvisual arrestins, arrestin-2 and arrestin-3, and determined the associated effects on the regulation of G protein-coupled receptor (GPCR) signaling. HEK293 cells stably expressing antisense constructs targeting arrestin-2 exhibited a selective reduction (approximately 50%) in arrestin-2 levels, while arrestin-3 antisense constructs resulted in reductions (>/=50%) in both arrestin-2 and arrestin-3 levels. Initial analysis of these cells demonstrated that a reduced level of arrestin expression resulted in a significant decrease in the extent of agonist-induced internalization of exogenously expressed beta2ARs, but had no effect on internalization of either m2 or m3 muscarinic acetylcholine receptors. Additional characterization involved assessing the role of arrestins in the regulation of endogenous GPCRs in these cells. Reduced arrestin levels significantly decreased the rate of endogenous beta2AR internalization, desensitization, and resensitization. Further analysis demonstrated that the desensitization of endogenous A2b adenosine and prostaglandin E2-stimulated receptors was also attenuated in cells with reduced arrestin levels. The effects on the beta2-adrenergic, A2b adenosine, and PGE2-stimulated receptors were similar among cell lines that exhibited either a selective reduction in arrestin-2 levels or a reduction in both arrestin-2 and -3 levels. These findings establish the utility of antisense approaches in the examination of arrestin-mediated GPCR regulation.

Adenylyl Cyclases↗
PubMed1999

Coupling of M2 muscarinic receptors to membrane ion channels via phosphoinositide 3-kinase gamma and atypical protein kinase C.

We report a novel signaling pathway linking M2 muscarinic receptors to metabotropic ion channels. Stimulation of heterologously expressed M2 receptors, but not other Gi/Go-associated receptors (M4 or alpha2c), activates a calcium- and voltage-independent chloride current in Xenopus oocytes. We show that the stimulatory pathway linking M2 receptors to these chloride channels consists of Gbeta gamma stimulation of phosphoinositide 3-kinase gamma (PI-3Kgamma), formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), and activation of atypical protein kinase C (PKC). The chloride current is activated in the absence of M2 receptor stimulation by the injection of PIP3, and PIP3 current activation is blocked by a pseudosubstrate inhibitory peptide of atypical PKC but not other PKCs. Moreover, the current is activated by injection of recombinant PKCzeta at concentrations as low as 1 nM. M2 receptor-current coupling was disrupted by inhibiton of PI-3K and by injection of beta gamma binding peptides, but it was not affected by expression of dominant negative p85 cRNA. We also show that this pathway mediates M2 receptor coupling to metabotropic nonselective cation channels in mammalian smooth muscle cells, thus demonstrating the broad relevance of this signaling cascade in neurotransmitter signaling.

Animals↗
PubMed1999

U50,488H-induced internalization of the human kappa opioid receptor involves a beta-arrestin- and dynamin-dependent mechanism. Kappa receptor internalization is not required for mitogen-activated protein kinase activation.

Agonist-promoted internalization of some G protein-coupled receptors has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internalization of the human and rat kappa opioid receptors stably expressed in Chinese hamster ovary cells, the potential mechanisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Exposure of the human kappa receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etorphine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat kappa opioid receptor. U50, 488H-induced human kappa receptor internalization was time- and concentration-dependent, with 30-40% of the receptors internalized following a 30-min exposure to 1 microM U50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50, 488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no effect on U50, 488H-induced internalization. In contrast, incubation with sucrose (0.4-0.8 M) significantly reduced U50,488H-induced internalization of the kappa receptor. While co-expression of the wild type GRK2, beta-arrestin, or dynamin I had no effect on kappa receptor internalization, co-expression of the dominant negative mutants GRK2-K220R, beta-arrestin (319-418), or dynamin I-K44A significantly inhibited receptor internalization. Whether receptor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant negative mutants of beta-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etorphine, which did not promote human kappa receptor internalization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat kappa receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internalization of the human kappa opioid receptor in Chinese hamster ovary cells occurs via a GRK-, beta-arrestin-, and dynamin I-dependent process that likely involves clathrin-coated pits. In addition, internalization of the kappa receptor is not required for activation of MAP kinase.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1999

Role of arrestins in endocytosis and signaling of alpha2-adrenergic receptor subtypes.

We investigated the role of arrestins in the trafficking of human alpha2-adrenergic receptors (alpha2-ARs) and the effect of receptor trafficking on p42/p44 MAP kinase activation. alpha2-ARs expressed in COS-1 cells demonstrated a modest level of agonist-mediated internalization, with alpha2c > alpha2b > alpha2a. However, upon coexpression of arrestin-2 (beta-arrestin-1) or arrestin-3 (beta-arrestin-2), internalization of the alpha2b AR was dramatically enhanced and redistribution of receptors to clathrin coated vesicles and endosomes was observed. Internalization of the alpha2c AR was selectively promoted by coexpression of arrestin-3, while alpha2a AR internalization was only slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any alpha2-AR subtype, either in the presence or absence of arrestins. Internalization of the alpha2b and alpha2c ARs was inhibited by coexpression of dominant negative dynamin-K44A. However, alpha2-AR-mediated activation of either endogenous or cotransfected p42/p44 mitogen-activated protein (MAP) kinase was not affected by either dynamin-K44A or arrestin-3. Moreover, activation of p42/p44 MAP kinase by endogenous epidermal growth factor, lysophosphatidic acid, and beta2-adrenergic receptors was also unaltered by dynamin-K44A. In summary, our data suggest that internalization of the alpha2b, alpha2c, and to a lesser extent alpha2a ARs, is both arrestin- and dynamin-dependent. However, endocytosis does not appear to be required for alpha2-adrenergic, epidermal growth factor, lysophosphatidic acid, or beta2-adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.

Amino Acid Sequence↗
PubMed1999

Regulation of G protein-coupled receptor kinases by caveolin.

G protein-coupled receptor kinases (GRKs) have been principally characterized by their ability to phosphorylate and desensitize G protein-coupled receptors. However, recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. Based on the identification of a consensus caveolin binding motif within the pleckstrin homology domain of GRK2, we tested the direct binding of purified full-length GRK2 to various glutathione S-transferase-caveolin-1 fusion proteins, and we discovered a specific interaction of GRK2 with the caveolin scaffolding domain. Interestingly, analysis of GRK1 and GRK5, which lack a pleckstrin homology domain, revealed in vitro binding properties similar to those of GRK2. Maltose-binding protein caveolin and glutathione S-transferase-GRK fusion proteins were used to map overlapping regions in the N termini of both GRK2 and GRK5 that appear to mediate conserved GRK/caveolin interactions. In vivo association of GRK2 and caveolin was suggested by co-fractionation of GRK2 with caveolin in A431 and NIH-3T3 cells and was further supported by co-immunoprecipitation of GRK2 and caveolin in COS-1 cells. Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides. These data reveal a novel mode for the regulation of GRKs that is likely to play an important role in their cellular function.

Amino Acid Sequence↗