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J L Benovic

Publications and source records attributed to J L Benovic.

At least 73 records · Page 4Linked to original sources
PubMed1998

Role of clathrin-mediated endocytosis in agonist-induced down-regulation of the beta2-adrenergic receptor.

Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the beta2-adrenergic receptor (beta2AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the beta2AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the beta2AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates beta2AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the beta2AR, suggesting that receptor internalization is critical for down-regulation in these cells. Moreover, a dominant-negative mutant of beta-arrestin, beta-arrestin-(319-418), also inhibits both agonist-induced receptor internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the beta2AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the beta2AR is in part due to trafficking of the beta2AR via the clathrin-coated pit endosomal pathway to lysosomes.

Adrenergic beta-Agonists↗
PubMed1998

Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged beta2-adrenergic receptor.

To date, the visualization of beta2-adrenergic receptor (beta2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a beta2AR conjugated with green fluorescent protein (beta2AR-GFP) provides the opportunity for a more extensive optical analysis of beta2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of beta2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of beta2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down-regulation), respectively. The observed temporal distribution of beta2AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different beta-agonists revealed time courses of beta2AR-GFP redistribution reflective of the intrinsic activity of each agonist.

Adrenergic beta-Agonists↗
PubMed1998

G-protein-coupled receptors: turn-ons and turn-offs.

Advances in the study of G-protein-coupled receptor regulation have provided novel insights into the role of G-protein-coupled receptor kinases and arrestins in this process. Of particular interest are recent studies that have dramatically expanded the known cellular functions of these molecules to include roles in receptor endocytosis and activation of MAP kinase signalling pathways.

Arrestins↗
PubMed1998

Enhanced expression of G protein-coupled receptor kinase 2 selectively increases the sensitivity of A2A adenosine receptors to agonist-induced desensitization.

1. G protein-coupled receptor kinases (GRKs) are thought to be important in mediating the agonist-induced phosphorylation and consequent desensitization of G protein-coupled receptor (GPCR) responses. We have previously shown that stable expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2) construct in NG108-15 mouse neuroblastoma x rat glioma cells suppresses the agonist-induced desensitization of A2A and A2B adenosine receptor-stimulated adenylyl cyclase activity (Mundell et al., 1997). To further determine the role of GRK2 in agonist-induced desensitization of these adenosine receptors, we stably overexpressed wild type GRK2 in NG108-15 cells. 2. In homogenates prepared from cells overexpressing GRK2, the acute stimulation of adenylyl cyclase by activation of A2A and A2B adenosine receptors was markedly reduced, but could be reversed by pretreating the cells with AD (adenosine deaminase), to remove extracellular adenosine from the medium. On the other hand, acute stimulation of adenylyl cyclase by secretin, iloprost, NaF and forskolin was the same in GRK2 overexpressing cells and plasmid-transfected control cells. 3. Cells overexpressing GRK2 were more sensitive to adenosine receptor agonist-induced desensitization than plasmid-transfected control cells. This effect was selective since the agonist sensitivity of desensitization for secretin and IP-prostanoid receptor-stimulated adenylyl cyclase activity was not affected by GRK2 overexpression. 4. These results further implicate GRK2 as the likely mechanism by which A2 adenosine receptors undergo short-term desensitization in NG108-15 cells, and indicate that even when overexpressed, GRK2 retains its substrate specificity for native receptors in intact cells. Furthermore, the susceptibility of GPCRs to desensitization appears to depend on the level of GRK expression, such that in cells that express high levels of GRK2, low agonist concentrations may be sufficient to trigger GRK-mediated desensitization.

Adenylyl Cyclases↗
PubMed1998

The role of receptor kinases and arrestins in G protein-coupled receptor regulation.

G protein-coupled receptors (GPRs) play a key role in controlling hormonal regulation of numerous second-messenger pathways. However, following agonist activation, most GPRs rapidly lose their ability to respond to hormone. For many GPRs, this process, commonly referred to as desensitization, appears to be primarily mediated by two protein families: G protein-coupled receptor kinases (GRKs) and arrestins. GRKs specifically bind to the agonist-occupied receptor, thereby promoting receptor phosphorylation, which in turn leads to arrestin binding. Arrestin binding precludes receptor/G protein interaction leading to functional desensitization. Many GPRs are then removed from the plasma membrane via clathrin-mediated endocytosis. Recent studies have implicated endocytosis in the resensitization of GPRs and have linked both GRKs and arrestins to this process. In this review, we discuss the role of GRKs and arrestins in regulating agonist-specific signaling and trafficking of GPRs.

Animals↗
PubMed1998

Mechanisms of acute desensitization of the beta2AR-adenylyl cyclase pathway in human airway smooth muscle.

beta2-Adrenergic receptors (beta2ARs) are important regulators of airway smooth muscle tone, and beta-sympathomimetic drugs are the most widely used agents in asthma therapy and are universally recognized as the treatment of choice for acute asthma attacks. Despite the clinical importance of beta-agonists and a good understanding of their mechanism of action in airway smooth muscle relaxation, surprisingly little is known about the manner in which the beta2AR signaling pathway is regulated in human airway smooth muscle (HASM). In this communication, we characterize mechanisms underlying rapid desensitization of the HASM beta2AR-adenylyl cyclase (AC) pathway. Acute homologous desensitization of beta2AR-mediated cyclic adenosine monophosphate (cAMP) production was characterized by an approximately 60% loss of maximal responsiveness to isoproterenol (ISO) when cells were pretreated for 30 min with 1 microM ISO. Acute heterologous beta2AR desensitization was characterized by an approximately 20% and 30% loss of maximal responsiveness to ISO challenge when cells were pretreated with forskolin and prostaglandin E2 (PGE2), respectively. Each form of desensitization was also characterized by an increase in the EC50 for ISO. beta2AR sequestration was associated with but not required for homologous desensitization. However, sequestration was required for rapid resensitization. Minimal alterations in inherent AC activity were observed with both modes of desensitization, suggesting that the beta2AR is the principal locus of regulation. Protein kinase inhibition by staurosporine largely reversed heterologous beta2AR desensitization and had a small but significant effect on homologous desensitization. In contrast, bisindolylmaleimide IX, a specific PKC-inhibitor, had no effect on heterologous or homologous beta2AR desensitization, suggesting that staurosporine effects were mediated by PKA inhibition. Overexpression of the G protein-coupled receptor kinase GRK2 in HASM cultures enhanced homologous desensitization. These data suggest that HASM beta2ARs are highly susceptible to rapid desensitization by multiple agents, and identify both GRKs and PKA as important mediators of acute beta2AR desensitization.

Adenylyl Cyclases↗
PubMed1997

Modulation of the arrestin-clathrin interaction in cells. Characterization of beta-arrestin dominant-negative mutants.

We recently demonstrated that nonvisual arrestins interact via a C-terminal binding domain with clathrin and function as adaptor proteins to promote beta2-adrenergic receptor (beta2AR) internalization. Here, we investigated the potential utility of a mini-gene expressing the clathrin-binding domain of beta-arrestin (beta-arrestin (319-418)) to function as a dominant-negative with respect to beta2AR internalization and compared its properties with those of beta-arrestin and beta-arrestin-V53D, a previously reported dominant-negative mutant. In vitro studies demonstrated that beta-arrestin-V53D bound better to clathrin than beta-arrestin but was significantly impaired in its interaction with phosphorylated G protein-coupled receptors. In contrast, whereas beta-arrestin (319-418) also bound well to clathrin it completely lacked receptor binding activity. When coexpressed with the beta2AR in HEK293 cells, beta-arrestin (319-418) effectively inhibited agonist-promoted receptor internalization, whereas beta-arrestin-V53D was only modestly effective. However, both constructs significantly inhibited the stimulation of beta2AR internalization by beta-arrestin in COS-1 cells. Interestingly, immunofluorescence microscopy analysis reveals that both beta-arrestin (319-418) and beta-arrestin-V53D are constitutively localized in clathrin-coated pits in COS-1 cells. These results indicate the potential usefulness of beta-arrestin (319-418) to effectively block arrestin-clathrin interaction in cells and suggest that this construct may prove useful in further defining the mechanisms involved in G protein-coupled receptor trafficking.

Animals↗
PubMed1997

Agonist-receptor-arrestin, an alternative ternary complex with high agonist affinity.

The rapid decrease of a response to a persistent stimulus, often termed desensitization, is a widespread biological phenomenon. Signal transduction by numerous G protein-coupled receptors appears to be terminated by a strikingly uniform two-step mechanism, most extensively characterized for the beta2-adrenergic receptor (beta2AR), m2 muscarinic cholinergic receptor (m2 mAChR), and rhodopsin. The model predicts that activated receptor is initially phosphorylated and then tightly binds an arrestin protein that effectively blocks further G protein interaction. Here we report that complexes of beta2AR-arrestin and m2 mAChR-arrestin have a higher affinity for agonists (but not antagonists) than do receptors not complexed with arrestin. The percentage of phosphorylated beta2AR in this high affinity state in the presence of full agonists varied with different arrestins and was enhanced by selective mutations in arrestins. The percentage of high affinity sites also was proportional to the intrinsic activity of an agonist, and the coefficient of proportionality varies for different arrestin proteins. Certain mutant arrestins can form these high affinity complexes with unphosphorylated receptors. Mutations that enhance formation of the agonist-receptor-arrestin complexes should provide useful tools for manipulating both the efficiency of signaling and rate and specificity of receptor internalization.

Adrenergic beta-Agonists↗
PubMed1997

Altered activity of palmitoylation-deficient and isoprenylated forms of the G protein-coupled receptor kinase GRK6.

G protein-coupled receptor kinases (GRKs) utilize diverse mechanisms to associate with the plasma membrane and mediate phosphorylation of agonist-occupied receptors. For example, two members of this family, GRK4 and GRK6, contain C-terminal cysteine residues that are palmitoylated. To address whether the activity and membrane association of GRK6 is regulated by palmitoylation, we overexpressed and characterized wild-type GRK6 and two GRK6 mutants, one with the palmitoylation sites mutated to serines (GRK6-pal-) and one containing a C-terminal CAAX motif to promote geranylgeranylation (GRK6-GG). Compared with wild-type GRK6, GRK6-pal- had a approximately 5-fold higher Km and approximately 2-fold lower Vmax for phosphorylating rhodopsin, whereas GRK6-GG exhibited a approximately 2-fold lower Km and approximately 14-fold higher Vmax for rhodopsin. In contrast, wild-type GRK6 and GRK6-pal- displayed similar activity toward the nonreceptor substrate phosvitin, indicating that nonpalmitoylated GRK6 is catalytically active. Wild-type GRK6 and GRK6-GG, but not GRK6-pal-, also bound significantly to phosphatidylcholine vesicles (36 +/- 3, 79 +/- 4, and 4 +/- 2%, respectively) suggesting that GRK6 activity is dependent upon its ability to interact with the plasma membrane. When assayed in COS-1 cells GRK6-pal- promoted minimal agonist-dependent sequestration of the beta2-adrenergic receptor, while sequestration was significantly increased in cells expressing either wild-type GRK6 or GRK6-GG. These data demonstrate an important functional link between the ability of GRK6 to bind to the plasma membrane, a process that appears to be regulated by palmitoylation, and its activity toward receptor substrates.

Amino Acid Sequence↗
PubMed1997

Internalization of the m2 muscarinic acetylcholine receptor. Arrestin-independent and -dependent pathways.

Recent studies have identified agonist-dependent phosphorylation as a critical event in the rapid uncoupling of the m2 muscarinic cholinergic receptors (mAChR) from G-proteins and sequestration of the receptors away from the cell surface. However, mutant m2 mAChRs were identified that were phosphorylated but unable to desensitize in adenylyl cyclase assays, while they internalized like wild type (WT) mAChRs. We have tested whether these properties might stem from differences in the abilities of the WT and mutant mAChR to bind arrestins, proteins implicated in both receptor/G-protein uncoupling and internalization. We have determined that arrestin binding requires phosphorylation at a cluster of Ser/Thr residues in amino acids 307-311 in the m2 mAChR. A strong correlation was found between the ability of WT and mutant receptors to bind arrestins in vitro or in vivo and to desensitize in adenylyl cyclase assays. However, the phosphorylation-dependent internalization of the m2 mAChR in HEK-tsA201 cells did not require arrestins and did not proceed via clathrin-mediated endocytosis. While the m2 mAChR was able to enter a clathrin- and arrestin-dependent pathway when arrestin 2 or arrestin 3 was significantly overexpressed, the preferred pathway of internalization of WT and certain mutant m2 mAChR in HEK-tsA201 cells did not involve participation of arrestins. The results suggest that the phosphorylation-mediated regulation of the m2 mAChR may involve arrestin-dependent and -independent events.

Animals↗
PubMed1997

Mechanism of quenching of phototransduction. Binding competition between arrestin and transducin for phosphorhodopsin.

Quenching of phototransduction in retinal rod cells involves phosphorylation of photoactivated rhodopsin by the enzyme rhodopsin kinase followed by binding of the protein arrestin. Although it has been proposed that the mechanism of arrestin quenching of visual transduction is via steric exclusion of transducin binding to phosphorylated light-activated rhodopsin (P-Rh*), direct evidence for this mechanism is lacking. In this study, we investigated both the role of rhodopsin phosphorylation in modulating its interaction with arrestin and transducin and the proposed binding competition between arrestin and transducin for P-Rh*. While the beta-adrenergic receptor kinase promotes significant arrestin binding to rhodopsin at a phosphorylation stoichiometry of >/=2 mol/mol, rhodopsin kinase promotes arrestin binding at a stoichiometry of approximately 0.9 mol/mol. Moreover, while beta-adrenergic receptor kinase phosphorylation of rhodopsin only modestly decreases transducin binding and activation, rhodopsin kinase phosphorylation of rhodopsin significantly decreases transducin binding and activation. Finally, arrestin competes effectively with transducin for binding to P-Rh* (50% inhibition at approximately 1:1 molar ratio of arrestin:transducin) but has no effect on transducin binding to nonphosphorylated light-activated rhodopsin (Rh*), paralleling the functional inhibition by arrestin on P-Rh*-stimulated transducin activation (50% inhibition at approximately 1.7:1 molar ratio of arrestin:transducin). These results demonstrate that a major role of rhodopsin phosphorylation is to promote high-affinity arrestin binding and decrease transducin binding thus allowing arrestin to effectively compete with transducin for binding to photoactivated rhodopsin.

Animals↗
PubMed1997

Regulation of G protein-coupled receptor kinases by calmodulin and localization of the calmodulin binding domain.

G protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple G protein-coupled receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Here we demonstrate that calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5 (IC50 approximately 50 nM) > GRK6 >> GRK2 (IC50 approximately 2 microM) >> GRK1. Calmodulin inhibition of GRK5 is mediated via a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Moreover, calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor even in the absence of calmodulin. Using glutathione S-transferase-GRK5 fusion proteins either to inhibit calmodulin-stimulated autophosphorylation or to bind directly to calmodulin, we determined that an amino-terminal domain of GRK5 (amino acids 20-39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues, characteristics typical of calmodulin binding sites, and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity.

Amino Acid Sequence↗
PubMed1997

Interaction of arrestins with intracellular domains of muscarinic and alpha2-adrenergic receptors.

The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein-coupled receptors as probes to screen brain cytosol proteins. Peptides from the third intracellular loop (i3) of the M2-muscarinic receptor (MR) (His208-Arg387), M3-MR (Gly308-Leu497), or alpha2A/D-adrenergic receptor (AR) (Lys224-Phe374) were generated in bacteria as glutathione S-transferase (GST) fusion proteins, bound to glutathione-Sepharose and used as affinity matrices to detect interacting proteins in fractionated bovine brain cytosol. Bound proteins were identified by immunoblotting following SDS-polyacrylamide gel electrophoresis. Brain arrestins bound to the GST-M3 fusion protein, but not to the control GST peptide or i3 peptides derived from the alpha2A/D-AR and M2-MR. However, each of the receptor subdomains bound purified beta-arrestin and arrestin-3. The interaction of the M3-MR and M2-MR i3 peptides with arrestins was further investigated. The M3-MR i3 peptide bound in vitro translated [3H]beta-arrestin and [3H]arrestin-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction of in vitro translated [3H]beta-arrestin, [3H]visual arrestin, and [3H]beta-arrestin/visual arrestin chimeras with the M2-MR i3 peptide were similar to those observed with the intact purified M2-MR that was phosphorylated and/or activated by agonist. Subsequent binding site localization studies indicated that the interaction of beta-arrestin with the M3-MR peptide required both the amino (Gly308-Leu368) and carboxyl portions (Lys425-Leu497) of the receptor subdomain. In contrast, the carboxyl region of the M3-MR i3 peptide was sufficient for its interaction with arrestin-3.

Animals↗
PubMed1997

Arrestin with a single amino acid substitution quenches light-activated rhodopsin in a phosphorylation-independent fashion.

Arrestins are members of a superfamily of regulatory proteins that participate in the termination of G protein-mediated signal transduction. In the phototransduction cascade of vertebrate rods, which serves as a prototypical G protein-mediated signaling pathway, the binding of visual arrestin is stimulated by phosphorylation of the C-terminus of photoactivated rhodopsin (Rh*). Arrestin is very selective toward light-activated phosphorhodopsin (P-Rh*). Previously we reported that a single amino acid substitution in arrestin, Arg175Gln, results in a dramatic increase in arrestin binding to Rh* [Gurevich, V. V., & Benovic, J. L. (1995) J. Biol. Chem. 270, 6010-6016]. Here we demonstrate that a similar mutant, arrestin(R175E), binds to light-activated rhodopsin independent of phosphorylation. Arrestin(R175E) binds with high affinity not only to P-Rh* and Rh* but also to light-activated truncated rhodopsin in which the C-terminus phosphorylation sites have been proteolytically removed. In an in vitro assay that monitored rhodopsin-dependent activation of cGMP phosphodiesterase (PDE), wild type arrestin quenched PDE response only when ATP was present to support rhodopsin phosphorylation. In contrast, as little as 30 nM arrestin(R175E) effectively quenched PDE activation in the absence of ATP. Arrestin(R175E) had no effect when the lifetime of Rh* no longer contributed to the time course of PDE activity, suggesting that it disrupts signal transduction at the level of rhodopsin-transducin interaction.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Arrestin/clathrin interaction. Localization of the clathrin binding domain of nonvisual arrestins to the carboxy terminus.

We have recently demonstrated that the nonvisual arrestins, beta-arrestin and arrestin3, interact with high affinity and stoichiometrically with clathrin, and we postulated that this interaction mediates internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this study, we localized the clathrin binding domain of arrestin3 using a variety of approaches. Truncation mutagenesis demonstrated that the COOH-terminal half of arrestin3 is required for clathrin interaction. Assessment of the clathrin binding properties of various glutathione S-transferase-arrestin3 fusion proteins indicated that the predominant clathrin binding domain is contained within residues 367-385. Alanine scanning mutagenesis further localized this domain to residues 371-379, and site-directed mutagenesis demonstrated the critical importance of both hydrophobic (Leu-373, Ile-374, and Phe-376) and acidic (Glu-375 and Glu-377) residues in the arrestin3/clathrin interaction. These results are complementary to the observation that hydrophobic and basic residues in clathrin are critical for its interaction with nonvisual arrestins (Goodman, O. B. , Jr., Krupnick, J. G., Gurevich, V. V., Benovic, J. L., and Keen, J. H. (1997) J. Biol. Chem. 272, 15017-15022). Lastly, an arrestin3 mutant in which Leu-373, Ile-374, and Phe-376 were mutated to Ala was significantly defective in its ability to promote beta2-adrenergic receptor internalization in COS-1 cells when compared with wild-type arrestin3. Taken together, these results implicate a discrete region of arrestin3 in high affinity binding to clathrin, an interaction critical for agonist-promoted internalization of the beta2-adrenergic receptor.

Alanine↗
PubMed1997

Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain.

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.

Alanine↗
PubMed1997

G-protein-coupled receptor kinase activity is increased in hypertension.

Impaired vascular beta-adrenergic responsiveness may play an important role in the development and/or maintenance of hypertension. This defect has been associated with an alteration in receptor/guanine nucleotide regulatory protein (G-protein) interactions. However, the locus of this defect remains unclear. G-Protein-coupled receptor kinases (GRKs) phosphorylate serine/threonine residues on G-protein-linked receptors in an agonist-dependent manner. GRK activation mediates reduced receptor responsiveness and impaired receptor/G-protein coupling. To determine whether the impairment in beta-adrenergic response in human hypertension might be associated with altered GRK activity, we studied lymphocytes from younger hypertensive subjects as compared with older and younger normotensive subjects. We assessed GRK activity by rhodopsin phosphorylation and GRK expression by immunoblot. GRK activity was significantly increased in lymphocytes from younger hypertensive subjects and paralleled an increase in GRK-2 (beta ARK-1) protein expression. In contrast, no alterations in cAMP-dependent kinase (A-kinase) activity or GRK-5/6 expression were noted. GRK activity was not increased in lymphocytes from older normotensive subjects who demonstrated a similar impairment in beta-adrenergic-mediated adenylyl cyclase activation. These studies indicate that GRK activity is selectively increased in lymphocytes from hypertensive subjects. The increase in GRK activity may underlie the reduction in beta-adrenergic responsiveness characteristic of the hypertensive state.

Adenylyl Cyclases↗