Biomedical subjects

J L Benovic

Publications and source records attributed to J L Benovic.

At least 55 records · Page 3Linked to original sources

PubMed1999

Internalization of the TXA2 receptor alpha and beta isoforms. Role of the differentially spliced cooh terminus in agonist-promoted receptor internalization.

Thromboxane A2 (TXA2) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction, atherosclerosis, and bronchial asthma. The TXA2 receptor (TXA2R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA2Rbeta, but not the TXA2Ralpha, undergoes agonist-induced internalization when expressed in HEK293 cells as well as several other cell types. Various dominant negative mutants were used to demonstrate that the internalization of the TXA2Rbeta is dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated pits in this process. Interestingly, the agonist-stimulated internalization of both the alpha and beta isoforms, but not of a mutant truncated after residue 328, can be promoted by overexpression of arrestin-3, identifying the C-tails of both receptors as necessary in arrestin-3 interaction. Simultaneous mutation of two dileucine motifs in the C-tail of TXA2Rbeta did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA2Rbeta is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA2R plays a critical role in regulating arrestin binding and subsequent receptor internalization.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1999

Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.

Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in beta-arrestin (arrestin2) and arrestin3 (beta-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported beta2-adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane.

Animals↗

PubMed1999

A direct role for arrestins in desensitization of the luteinizing hormone/choriogonadotropin receptor in porcine ovarian follicular membranes.

The luteinizing hormone/choriogonadotropin (LH/CG) receptor (R) is a heptahelical R that, upon agonist binding, activates the stimulatory guanine nucleotide-binding protein (Gs) and the downstream effector adenylyl cyclase (AC). Like other G protein-coupled Rs, the LH/CG R subsequently exhibits reduced agonist-dependent effector activity, or desensitization, in response to saturating agonist. Unlike desensitization of many other G protein-coupled Rs, the in vivo desensitization response of LH/CG R-stimulated AC activity of ovarian follicles to the preovulatory surge of LH can be mimicked under cell-free conditions. Based on evidence that porcine ovarian follicular membranes unexpectedly contained beta-arrestin-1, the role of arrestins in desensitization of the LH/CG R was investigated. Results showed that neutralizing arrestin antibodies blocked the development of desensitization and that desensitization was rescued with a synthetic peptide corresponding to the antibody-binding epitope on beta-arrestin-1. These results suggest that endogenous beta-arrestin-1 participates in agonist-dependent desensitization of the LH/CG R. Addition of recombinant purified beta-arrestin-1 mimicked human chorionic gonadotrophin to promote desensitization of human chorionic gonadotrophin-stimulated AC activity, in the presence of the ATP phosphorylation antagonist adenylyl-imidodiphosphate, with an ED50 of approximately 0.1 nM. Increased levels of an 87-kDa protein reactive with glycoprotein hormone R-reactive antibody, consistent with the LH/CG R, coimmunoprecipitated with follicular membrane beta-arrestin-1 in response to LH/CG R activation compared with unactivated R. Taken together, these results show that ovarian follicles contain membrane-associated beta-arrestin-1, that beta-arrestin-1 participates in agonist-dependent desensitization of the LH/CG R, and that the trigger for beta-arrestin-1 binding to the LH/CG R appears to be R activation.

Adenylyl Imidodiphosphate↗

PubMed1999

G-protein-coupled receptor kinase expression in hypertension.

In human hypertension we have recently identified an increase in lymphocyte G-protein receptor kinase-2 (GRK-2) protein expression, the key protein regulating the interaction between G-protein-coupled receptors and activation of adenylyl cyclase. However, it was not known whether this increase in GRK-2 protein expression was attributable to regulation at the level of translation. Furthermore, the relationship between extent of GRK-2 expression, receptor activation of adenylyl cyclase, and blood pressure was unclear. We therefore studied lymphocytes from 7 young subjects with borderline hypertension and 14 young normotensive subjects. Immunodetectable GRK-2 protein expression in lymphocytes from subjects with hypertension was increased (155%+/-7% of normotensive subjects; P < .05). In addition, GRK-2 protein expression was positively correlated with blood pressure (r = 0.53; P = .013) and inversely correlated with beta-adrenergic-mediated adenylyl cyclase activity (r = -0.54, P = .012). However, lymphocyte GRK-2 messenger ribonucleic acid (mRNA) content was not altered (110%+/-13% of that observed in normotensive control subjects). Increased GRK-2 protein expression may be an important factor in the impairment of beta-adrenergic-mediated vasodilation, characteristic of the hypertensive state.

Adult↗

PubMed1999

Molecular events associated with the regulation of signaling by M2 muscarinic receptors.

Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs.

Adenylate Cyclase Toxin↗

PubMed1999

Selective reduction in A2 adenosine receptor desensitization following antisense-induced suppression of G protein-coupled receptor kinase 2 expression.

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is considered to play a critical role in the desensitization of responses mediated by these receptors. To explore the role of GRK2 in A2 adenosine receptor desensitization, we attempted to reduce specifically GRK2 expression in NG108-15 cells by stable transfection with an antisense rat GRK2 cDNA sequence. This yielded up to a 69% loss of GRK2 when compared with plasmid-transfected control cells, which correlated with a reduction in kinase activity when measured by the ability of cell lysates to promote light-dependent phosphorylation of rhodopsin. Levels of GRK3 were the same in antisense and plasmid-transfected controls. On addition of the A2 adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine, cyclic AMP accumulation was greater in GRK2 antisense cells as compared with plasmid control cells. In contrast, cyclic AMP accumulation via agonist stimulation of either IP-prostanoid or secretin receptors or by addition of forskolin was not significantly different among all clones examined. The increase in A2 adenosine receptor response could not be explained by changes in A2A adenosine receptor expression, as assessed by ligand binding experiments with the radioligand 2-3H-labelled 4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-++ +ylamino]ethyl]phenol ([3H]ZM241385). These data show for the first time a direct correlation between expression of GRK2 and desensitization of natively expressed A2 adenosine receptors in intact cells, suggesting that GRK2 plays a major role in the regulation of these receptors. Key Words: G protein-coupled receptor kinase-G protein-coupled receptor-Antisense-NG108-15 cells-A2 adenosine receptors-Desensitization.

Animals↗

PubMed1999

MAPK superfamily activation in human airway smooth muscle: mitogenesis requires prolonged p42/p44 activation.

Asthma is frequently associated with abnormal airway smooth muscle (ASM) growth that may contribute to airway narrowing and hyperresponsiveness to contractile agents. Although numerous hormones and cytokines have been shown to induce human ASM (HASM) proliferation, the cellular and molecular mechanisms underlying HASM hyperplasia are largely unknown. Here we characterize the roles of the mitogen-activated protein kinase (MAPK) superfamily [p42/p44 MAPK, c-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38] in mediating hormone- and cytokine-induced HASM proliferation. Significant enhancement of [(3)H]thymidine incorporation in HASM cultures was observed only by treatment with agents (epidermal growth factor, platelet-derived growth factor, thrombin, and phorbol 12-myristate 13-acetate) that promoted a strong and sustained activation of p42/p44 MAPK. Significant activation of the JNK/SAPK and p38 pathways was only observed on stimulation with interleukin (IL)-1beta and tumor necrosis factor-alpha, agents that did not appreciably stimulate HASM proliferation. Two different inhibitors of MAPK/extracellular signal-regulated kinase kinase (MEK), PD-98059 and U-0126, inhibited mitogen-induced [3H]thymidine incorporation in a manner consistent with their ability to inhibit p42/p44 activation. Elk-1 and activator protein-1 reporter activation by mitogens was similarly inhibited by inhibition of MEK, suggesting a linkage between p42/p44 activation, transcription factor activation, and HASM proliferation. These findings establish a fundamental role for p42/p44 activation in regulating HASM proliferation and provide insight into species-specific differences observed among studies in ASM mitogenesis.

Cell Division↗

PubMed1999

Inflammatory and contractile agents sensitize specific adenylyl cyclase isoforms in human airway smooth muscle.

Beta-agonists, through activation of the beta(2)-adrenergic receptor (beta(2)AR)-G(s)-adenylyl cyclase (AC) pathway, promote bronchodilation via functional antagonism of airway smooth muscle (ASM) spasmogens associated with the asthmatic state. Although previous studies have demonstrated that beta(2)AR signaling in ASM is subject to homologous (beta-agonist-induced) beta(2)AR desensitization, the potential for inflammatory and contractile agents to impact beta(2)AR signaling in ASM through heterologous mechanisms has not been defined. Here we report that chronic exposure of human ASM (HASM) to carbachol, serotonin, the thromboxane analogue U46619, or histamine induced little change or a small increase in isoproterenol-stimulated cyclic adenosine monophosphate (cAMP) formation, but significantly increased cAMP formation elicited by stimulation with forskolin. This latter increase in intrinsic AC activity was largely reversed by pertussis toxin pretreatment, and was unaffected by protein kinase C inhibition. Analysis of both AC function and isoform expression supports a dominant role of AC VI in HASM, and points to important differences in ASM AC isoform expression among species. Additional studies identify AC as the limiting component in beta(2)AR-G(s)-AC signaling in HASM, and thus a potentially important target of therapeutic strategies designed to influence airway contractile state.

Adenylyl Cyclases↗

PubMed1999

Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor.

The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4alpha, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or G alpha t, a scavenger of G betagamma. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and G alpha t inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.

Animals↗

PubMed1999

Pharmacological inhibition of protein kinases in intact cells: antagonism of beta adrenergic receptor ligand binding by H-89 reveals limitations of usefulness.

The use of pharmacological inhibitors of protein kinases represents a potentially powerful tool in dissecting the regulatory features of intracellular signaling pathways. However, although the in vitro potency, selectivity, and efficacy of numerous kinase inhibitors have been characterized, little is known regarding the usefulness of these compounds as inhibitors in intact cells. In attempting to characterize the role of protein kinase A (PKA) in regulating the beta-2 adrenergic receptor (AR) in human airway cells, we observed a seemingly profound capacity of the isoquinoline H-89, a potent and widely used PKA inhibitor, to attenuate agonist-mediated desensitization of the beta-2 AR. Although additional experiments identified H-89 as an effective inhibitor of intracellular PKA, extended analysis of the compound determined the principal effect of H-89 was via its action as a beta-2 AR antagonist. Pretreatment with or the acute addition of H-89 significantly attenuated isoproterenol-stimulated cAMP accumulation. In cells pretreated with H-89 and then washed extensively, the subsequent dose-dependent response to isoproterenol suggested beta-2 AR antagonism by retained H-89. Competition binding of [125I]iodopindolol established Ki values of approximately 180 nM and 350 nM for H-89 antagonism of beta-2 AR and beta-1 AR, respectively. Additional receptor binding studies suggest selectivity of H-89 for the beta-2 AR and beta-1 AR, although a weak antagonism (Ki values of approximately 10 microM or greater) of other G protein-coupled receptors was observed. Results from additional pharmacological and biochemical analyses of various protein kinase inhibitors further established the need for careful characterization of pharmacological inhibitors when used in intact cell models.

Adrenergic beta-Antagonists↗

PubMed1998

Characterization of dominant negative arrestins that inhibit beta2-adrenergic receptor internalization by distinct mechanisms.

Arrestins have been shown to act as adaptor proteins that mediate the interaction of G protein-coupled receptors with the endocytic machinery. In this study, the role of arrestin-3 in receptor internalization was investigated by constructing different arrestin-3 minigenes that could potentially act as dominant negative inhibitors of arrestin function. Expression of arrestin-3 proteins containing amino acids 1-320 or 201-409 resulted in the inhibition of beta2-adrenergic receptor internalization in HEK-293 cells by approximately 40%. Both of these arrestins were diffusely localized within the cytoplasm of transfected cells, were unable to mediate redistribution of receptors to clathrin-coated pits, and did not localize to coated pits in either the presence or absence of receptor and agonist. Arrestin-3(1-320), but not arrestin-3(201-409), bound to light-activated phosphorylated rhodopsin with an affinity comparable with that of wild-type arrestin-3. In contrast, expression of arrestin-3 proteins composed of only the clathrin binding domain, arrestin-3(284-409), and arrestin-3(290-409) resulted in the constitutive localization of these arrestins to coated pits. Arrestin-3(284-409) and arrestin-3(290-409) acted as dominant negative inhibitors of wild-type arrestin function, inhibiting receptor internalization by 70 and 30%, respectively. Carboxyl-terminal deletions of arrestin-3 retained the ability to promote internalization until residues amino-terminal to amino acid 350 were deleted, suggesting that residues in this region also compose part of the clathrin binding domain in addition to the major binding site between residues 371-379. These studies characterize at least two distinct mechanisms, competition for either receptor or clathrin binding, by which dominant negative arrestins inhibit receptor internalization and further define residues within arrestin-3 that constitute the clathrin binding domain.

Adrenergic beta-2 Receptor Antagonists↗

PubMed1998

Structure-function analysis of G protein-coupled receptor kinase-5. Role of the carboxyl terminus in kinase regulation.

Many G protein-coupled receptors are phosphorylated and regulated by a distinct family of G protein-coupled receptor kinases (GRKs) that specifically target the activated form of the receptor. Recent studies have revealed that the GRKs are also subject to post-translational regulation. For example, GRK5 activity is strongly inhibited by protein kinase C phosphorylation and by Ca2+-calmodulin binding. Ca2+-calmodulin binding also promotes GRK5 autophosphorylation, which further contributes to kinase inhibition. In this study we identify two important structural domains in GRK5, a phospholipid binding domain (residues 552-562) and an autoinhibitory domain (residues 563-590), that significantly contribute to GRK5 localization and function. We demonstrate that the C-terminal region of GRK5 (residues 563-590) contains residues autophosphorylated in the presence of calmodulin as well as the residues phosphorylated by protein kinase C. Deletion of this domain increases the apparent affinity of GRK5 for receptor substrates 3-4-fold but has no effect on nonreceptor substrates. These findings define residues 563-590 of GRK5 as an autoinhibitory domain with efficacy that is regulated by phosphorylation. Another C-terminal domain in GRK5 that appears to be functionally important is found between residues 552 and 562. Deletion of this region significantly inhibits kinase phosphorylation of membrane-bound receptor substrates but has no effect on soluble substrates. Additional studies reveal that this domain is critical for GRK5 interaction with phospholipids and for the intracellular localization of the kinase. Interestingly, similar regions in GRK4 and GRK6 appear to be palmitoylated (and involved in membrane interaction), suggesting evolutionary conservation of the function of this domain.

Amino Acid Sequence↗

PubMed1998

Localization of the sites for Ca2+-binding proteins on G protein-coupled receptor kinases.

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins has recently emerged as a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+-calmodulin. To dissect the mechanism of this inhibition at the molecular level, we localized the GRK domains involved in Ca2+-binding protein interaction using a series of GST-GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5-calmodulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuated the effect of calmodulin on GRK5, and the simultaneous mutation of both sites rendered the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca2+-dependent regulation of GRKs.

Amino Acid Sequence↗

PubMed1998

Signaling and phosphorylation-impaired mutants of the rat follitropin receptor reveal an activation- and phosphorylation-independent but arrestin-dependent pathway for internalization.

We have previously shown that the rat follitropin receptor (rFSHR) expressed in transfected cells becomes phosphorylated upon stimulation of the cells with agonist or a phorbol ester. Peptide mapping and mutagenesis studies have also shown that the agonist- or phorbol ester-induced phosphorylation of the rFSHR maps to Ser/Thr residues present in the first and third intracellular loops. The experiments presented herein were initially designed to test for the presence of additional phosphorylation sites on the second intracellular loop of the rFSHR. Analysis of two new mutants in which the two threonines in the second intracellular loop (rFSHR-2L) or the two threonines in the second intracellular loop and the seven Ser/Thr residues in the third intracellular loop (rFSHR-2L + 3L) were mutated showed that one or more of the two threonines in the second intracellular loop are phosphorylated in response to phorbol ester, but not in response to agonist stimulation. Since rFSHR-2L and rFSHR-2L + 3L displayed a reduction in agonist-induced signaling, two additional mutants (rFSHR-D389N and rFSHR-Y530F) were constructed in an attempt to better understand the relationship between the agonist-induced activation, phosphorylation, and internalization of the rFSHR. These point mutations impaired agonist-stimulated signal transduction and abolished agonist-induced phosphorylation. Co-transfection studies revealed that the phosphorylation of these mutants can be rescued by overexpression of G protein-coupled receptor kinase 2, but this increased phosphorylation only rescues the internalization of rFSHR-D389N. The internalization of both mutants could be rescued by overexpression of arrestin-3, however. Taken together, these results argue that the agonist-induced activation and phosphorylation of the rFSHR are not essential for internalization. while the interaction of the rFSHR with a nonvisual arrestin is essential for internalization.

Amino Acid Substitution↗

PubMed1998

Binding and phosphorylation of tubulin by G protein-coupled receptor kinases.

Although the beta-adrenergic receptor kinase (betaARK) mediates agonist-dependent phosphorylation and desensitization of G protein-coupled receptors, recent studies suggest additional cellular functions. During our attempts to identify novel betaARK interacting proteins, we found that the cytoskeletal protein tubulin could specifically bind to a betaARK-coupled affinity column. In vitro analysis demonstrated that betaARK and G protein-coupled receptor kinase-5 (GRK5) were able to stoichiometrically phosphorylate purified tubulin dimers with a preference for beta-tubulin and, under certain conditions, the betaIII-isotype. Examination of the GRK/tubulin binding characteristics revealed that tubulin dimers and assembled microtubules bind GRKs, whereas the catalytic domain of betaARK contains the primary tubulin binding determinants. In vivo interaction of GRK and tubulin was suggested by the following: (i) co-purification of betaARK with tubulin from brain tissue; (ii) co-immunoprecipitation of betaARK and tubulin from COS-1 cells; and (iii) co-localization of betaARK and GRK5 with microtubule structures in COS-1 cells. In addition, GRK-phosphorylated tubulin was found preferentially associated with the microtubule fraction during in vitro assembly assays suggesting potential functional significance. These results suggest a novel link between the cytoskeleton and GRKs that may be important for regulating GRK and/or tubulin function.

Animals↗

PubMed1998

Mutation of individual serine residues in the C-terminal tail of the lutropin/choriogonadotropin receptor reveal distinct structural requirements for agonist-induced uncoupling and agonist-induced internalization.

We have previously mapped the agonist-induced phosphorylation of the rat lutropin/choriogonadotropin receptor (rLHR) to a locus of four serines (Ser635, Ser639, Ser649, and Ser652) located in the C-terminal tail. The removal or mutation of this locus delays the time course of agonist-induced uncoupling of the rLHR from its effector system without affecting the overall magnitude of uncoupling, and it retards the endocytosis of the agonist-receptor complex. We have now prepared and analyzed four new rLHR mutants in which each of these serines were individually mutated to alanines. The data presented show that each mutation reduces agonist-promoted rLHR phosphorylation by 20-40%. Mutation of Ser635 or Ser639 delayed the time course of agonist-induced uncoupling to about the same extent as the simultaneous mutation of all four serines. Mutation of Ser635 or Ser639 also retarded agonist-induced internalization, but the magnitude of this decrease was less than that induced by the simultaneous mutation of all four serines. Mutation of Ser649 had no effect on agonist-induced uncoupling but retarded agonist-induced internalization to the same extent as the simultaneous mutation of all four serines. Mutation of Ser652 has little or no effect on either of these two parameters. Co-transfection studies with dominant-negative arrestins and dominant-negative dynamin reveal that, despite differences in their rates of internalization, rLHR-wild-type, rLHR-S639A, and rLHR-S649A are internalized by an arrestin- and dynamin-dependent pathway. These data show that the structural requirements needed for the agonist-induced uncoupling and internalization of the rLHR are distinct.

Alanine↗

PubMed1998

Arrestin-independent internalization of the m1, m3, and m4 subtypes of muscarinic cholinergic receptors.

To understand what processes contribute to the agonist-induced internalization of subtypes of muscarinic acetylcholine receptors, we analyzed the role of arrestins. Whereas the m2 mAChR has been shown to undergo augmented internalization when arrestins 2 and 3 are overexpressed (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-23689), the agonist-induced internalization of m1, m3, and m4 mAChRs was unchanged when arrestins 2 or 3 were overexpressed in transiently transfected HEK-tsA201 cells. Furthermore, when a dominant-negative arrestin was used to interrupt endogenous arrestin function, there was no change in the internalization of the m1, m3, and m4 mAChR whereas the internalization of the beta2 adrenergic receptor was completely blocked. Wild-type and GTPase-deficient dominant-negative dynamin were used to determine which endocytic machinery played a role in the endocytosis of the subtypes of mAChRs. Interestingly, when dynamin function was blocked by overexpression of the GTPase-deficient dynamin, agonist- induced internalization of the the m1, m3, and m4 mAChRs was suppressed. These results suggested that the internalization of the m1, m3, and m4 mAChRs occurs via an arrestin-independent but dynamin-dependent pathway. To ascertain whether domains that confer arrestin sensitivity and dynamin insensitivity could be functionally exchanged between subtypes of mAChRs, chimeric m2/m3 receptors were analyzed for their properties of agonist-induced internalization. The results demonstrated that the third intracellular loop of the m2 mAChR conferred arrestin sensitivity and dynamin insensitivity to the arrestin-insensitive, dynamin-sensitive m3 mAChR while the analogous domain of the m3 mAChR conferred arrestin resistance and dynamin sensitivity to the previously arrestin-sensitive, dynamin-insensitive m2 mAChR.

Arrestin↗

PubMed1998

Receptor docking sites for G-protein betagamma subunits. Implications for signal regulation.

We report the direct interaction of Gbetagamma with the third intracellular (i3) loop of the M2- and M3-muscarinic receptors (MR) and the importance of this interaction relative to effective phosphorylation of the receptor subdomain. The i3 loop of the M2- and the M3-MR were expressed in bacteria and purified as glutathione S-transferase fusion proteins for utilization as an affinity matrix and to generate substrate for receptor subdomain phosphorylation. In its inactive heterotrimeric state stabilized by GDP, brain G-protein did not associate with the i3 peptide affinity matrix. However, stimulation of subunit dissociation by GTPgammaS/Mg2+ resulted in the retention of Gbetagamma, but not the Galpha subunit, by the M2- and M3-MR i3 peptide resin. Purified Gbetagamma bound to the M3-MR i3 peptide with an apparent affinity similar to that observed for the Gbetagamma binding domain of the receptor kinase GRK2 and Bruton tyrosine kinase, whereas transducin betagamma was not recognized by the M3-MR i3 peptide. Effective phosphorylation of the M3-MR peptide by GRK2 required both Gbetagamma and lipid as is the case for the intact receptor. Incubation of purified GRK2 with the i3 peptide in the presence of Gbetagamma resulted in the formation of a functional ternary complex in which Gbetagamma served as an adapter protein. Such a complex provides a mechanism for specific spatial translocation of GRK2 within the cell positioning the enzyme on its substrate, the activated receptor. The apparent ability of Gbetagamma to act as a docking protein may also serve to provide an interface for this class of membrane-bound receptors to an expanded array of signaling pathways.

Animals↗