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J L Benovic

Publications and source records attributed to J L Benovic.

At least 91 records · Page 5Linked to original sources
PubMed1997

Regulation of the G protein-coupled receptor kinase GRK5 by protein kinase C.

G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the hormone-occupied form of numerous G protein-coupled receptors, ultimately resulting in termination of receptor signaling. While little is presently known about the regulation of GRK function, recent studies suggest a role for protein kinase C (PKC) phosphorylation of the beta-adrenergic receptor kinase in membrane association and activation of the kinase. To assess a potential general role for PKC in regulating GRK function, we characterized the ability of PKC to phosphorylate GRK5, a recently identified member of the GRK family. We demonstrate that GRK5 can be rapidly and stoichiometrically phosphorylated by PKC in vitro. Intact cell studies reveal that GRK5 is also phosphorylated when transiently expressed in COS-1 cells following treatment with the PKC activator, phorbol 12-myristate 13-acetate. In vitro analysis reveals two major sites of PKC phosphorylation within the C-terminal 26 amino acids of GRK5. GRK5 phosphorylation by PKC dramatically reduces its ability to phosphorylate both receptor (light-activated rhodopsin) and non-receptor (casein and phosvitin) substrates. Kinetic analysis reveals an approximately 5-fold increased Km and approximately 3-fold decreased Vmax for rhodopsin, with no change in the Km for ATP. The reduced affinity of PKC-phosphorylated GRK5 for rhodopsin was also evident in a decreased ability to bind to rhodopsin-containing membranes, while direct binding of GRK5 to phospholipids appeared unaltered. These results suggest that PKC might play an important role in modulating the ability of GRK5 to regulate receptor signaling and that GRK phosphorylation by PKC may serve as a disparate mechanism for regulating GRK activity.

Amino Acid Sequence↗
PubMed1997

Identification and chromosomal localization of a processed pseudogene of human GRK6.

G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied G-protein-coupled receptors, resulting in desensitization of receptor signaling. To date, 6 mammalian GRKs have been identified by molecular cloning. Several lines of evidence indicate that a homologue of GRK6, the most recently described GRK, is present in the human genome. Northern analysis identifies two transcripts which hybridize to GRK6, and genomic Southern analysis indicates that GRK6 is localized to chromosome 5, with a second GRK6-like locus on chromosome 13. To identify the GRK6 homologue on chromsome 13, several sets of closely-spaced primers were designed based on the GRK6 cDNA sequence and then used to amplify human genomic DNA by PCR. Two products were identified, the larger of which is a fragment of the GRK6 gene which contains introns, while the smaller fragment is 94% homologous to GRK6 and contains no introns. In order to further characterize this GRK6 homologue, primers from the 5' and 3' coding regions of GRK6 were used to amplify a product of 1458 base pairs from human genomic DNA. This 1458 base pair PCR fragment displays 94% homology to GRK6 and contains multiple nucleotide insertions and deletions compared to GRK6, including a C to T mutation at base pair 202 which creates a predicted in-frame stop codon. In an effort to determine whether this gene is transcriptionally active, primers designed to preferentially amplify either GRK6 or the homologue were used in reverse transcription PCR. In contrast to the GRK6-specific primers, primers which selectively amplify the GRK6 homologue fail to produce a PCR product in any RNA tested, indicating that this gene is most likely transcriptionally inactive. PCR amplification of rodent/human hybrid cell lines using these same primers confirms the previously established chromosome 5 localization of GRK6, and localizes this homologue to chromosome 13. Northern analysis indicates that the two GRK6-hybridizing species seen in RNA differ by approximately 500 base pairs in the 3' untranslated region, indicating that both transcripts likely arise from differential processing of a single gene. Taken together, these data indicate that the GRK6-hybridizing species on chromosome 13 is a transcriptionally inactive processed pseudogene of GRK6, while the two GRK6 transcripts differ in the 3' untranslated region.

Base Sequence↗
PubMed1997

Beta-adrenergic receptor kinase-1 acutely regulates PTH/PTHrP receptor signalling in human osteoblastlike cells.

To investigate whether G protein-coupled receptor kinases (GRKs) are involved in the regulation of the PTH/PTHrPR, we have established mutant SaOS-2 cells which stably overexpress (> 10-20-fold) a dominant negative form of the beta-adrenergic receptor kinase-1 (beta ARK-1). Acute (< or = 2 h) incubation with hPTH (1-34) induced significantly less (by up to 50%) downregulation of the PTH/PTHrPR in beta ARK-1 mutant SaOS-2 cells than observed in wild-type cells. Pretreatment of wild-type cells with PTH for 2 h induced homologous cAMP desensitisation to a second challenge with PTH, while the effect was blunted by up to 60% in beta ARK-1 mutant cells. We conclude that activation of beta ARK-1 (or a closely related GRK) is a critical component of the acute phase (< or = 2 h) of PTH-induced receptor downregulation and homologous cAMP desensitisation of the PTH/PTHrPR.

Cell Line↗
PubMed1997

Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding state.

Arrestin plays an important role in quenching phototransduction via its ability to interact specifically with the phosphorylated light-activated form of the visual receptor rhodopsin (P-Rh*). Previous studies have demonstrated that Arg175 in bovine arrestin is directly involved in the phosphorylation-dependent binding of arrestin to rhodopsin and seems to function as a phosphorylation-sensitive trigger. In this study, we further probed the molecular mechanism of phosphorylation recognition by substituting 19 different amino acids for Arg175. We also assessed the effects of mutagenesis of several other highly conserved residues within the phosphorylation-recognition region (Val170, Leu172, Leu173, Ile174, Val177, and Gln178). The binding of all of these mutants to P-Rh*, light-activated rhodopsin, and truncated rhodopsin, which lacks the carboxyl-terminal phosphorylation sites, was then characterized. Overall, our results suggest that arrestin interaction with the phosphorylated carboxyl-terminal domain of rhodopsin activates two relatively independent changes in arrestin: (a) mobilization of additional binding sites and (b) increased affinity of the phosphorylation-recognition region for the rhodopsin carboxyl-terminal domain. Together, these two mechanisms ensure the exquisite selectivity of arrestin toward P-Rh*. Mutagenesis of residues that play a major role in binding site mobilization and phosphorylation-recognition enabled us to create "constitutively active" (phosphorylation-independent) arrestin mutants that have high affinity for both P-Rh* and light-activated rhodopsin. The introduction of a negative charge in position 175 was particularly effective in this respect. A detailed molecular model of phosphorylation-recognition is proposed.

Amino Acid Sequence↗
PubMed1997

A dominant negative mutant of the G protein-coupled receptor kinase 2 selectively attenuates adenosine A2 receptor desensitization.

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 responses. NG108-15 mouse neuroblastoma X rat glioma cells express a wide range of G protein-coupled receptors and significant levels of GRK2. Therefore, to determine the role of GRK2 in agonist-induced desensitization of various G(s)-coupled receptors in NG108-15 cells, we stably transfected cells with a dominant negative mutant GRK2 construct (Lys220Arg). In homogenates prepared from cells overexpressing the dominant negative mutant GRK2, the acute stimulation of adenylyl cyclase by various receptor and nonreceptor agonists was the same as in control cells stably transfected with plasmid only. NG108-15 cells express both A2a and A2b adenosine receptors, which mediate activation of adenylyl cyclase, with both of these responses being subject to agonist-induced desensitization with a t1/2 of 15-20 min. In dominant negative mutant GRK2 cells, the rates of desensitization of A2a and A2b receptor-stimulated adenylyl cyclase were markedly slower than in plasmid transfected controls, with the latter being similar to wild-type cells. After a 20-min treatment with an adenosine agonist, the desensitization of A2a and A2b receptor-stimulated adenylyl cyclase in dominant negative mutant GRK2 cells was less than half that seen in plasmid transfected control cells. On the other hand, the agonist-induced desensitization of secretin and IP-prostanoid receptor-stimulated adenylyl cyclase was the same in dominant negative mutant GRK2 cells as in plasmid transfected control cells. These results indicate that in intact cells, GRK2 may mediate the desensitization of adenosine A2 receptors. Furthermore, there seems to be selectivity of GRK2 action between G(s)-coupled receptors because the agonist-induced desensitization of secretin and IP-prostanoid receptor-stimulated adenylyl cyclase was not affected by dominant negative mutant GRK2 overexpression.

Adenosine↗
PubMed1997

Chronic effects of catecholamines on the beta 2-adrenoreceptor system in cultured human airway epithelial cells.

Chronic catecholamine treatment induces beta-adrenergic receptor (beta AR) downregulation, i.e., a loss of total cell receptors. In the human respiratory tract, the mechanism(s) underlying beta AR downregulation remains poorly understood. The present study, therefore, examined the effects of 24 h of exposure to isoproterenol (Iso; 10 nM or 1 microM) on beta AR density and the rate of beta AR degradation, steady-state beta 2-adrenergic receptor (beta 2 AR) mRNA levels, and the content of Gs alpha and Gi alpha proteins in cultured human bronchial epithelial cells (i.e., the BEAS-2B cell line). beta AR density assessed by binding with [125I]iodopindolol decreased in a dose-dependent fashion with 24 h of Iso exposure. With Iso (1 microM), beta AR density decreased by approximately 82%. In contrast, forskolin (100 microM) and dibutyryl adenosine 3',5'-cyclic monophosphate (1 mM), agents that also increase adenosine 3',5'-cyclic monophosphate (cAMP) levels, had no significant effect on beta AR density. Iso exposure also elicited a concomitant decrease in Iso-stimulated cAMP but had no significant effect on the content of the G proteins G alpha i2 and Gs alpha assessed by immunoblotting and toxin-catalyzed ADP ribosylation. Of note, Iso exposure (1 microM) had no effect on steady-state levels of beta 2 AR mRNA measured both by Northern analysis and by reverse transcriptase-polymerase chain reaction. However, beta AR half-life assessed in the presence of the protein synthesis inhibitor cycloheximide was reduced by approximately 60% in Iso-treated cells (i.e., from 37 h in control to 16 h in 1 microM Iso). These results suggest that, in human airway epithelial cells, beta 2 AR downregulation 1) is not primarily driven by intracellular cAMP levels, 2) is not associated with significant decreases in steady-state levels of beta 2 AR mRNA, and 3) is largely posttranslationally regulated by increases in the rate of receptor protein degradation.

Adrenergic beta-Agonists↗
PubMed1996

Differentially regulated expression of the G-protein-coupled receptor kinases, betaARK and GRK6, during myelomonocytic cell development in vitro.

G-protein-coupled receptor kinases (GRKs) mediate agonist-specific phosphorylation and desensitization of G-protein-coupled receptors. Previous studies have shown that several of these kinases are expressed in hematopoietic cells and that GRK expression is modulated during T-lymphocyte activation. Here, we analyzed the regulation of beta-adrenergic receptor kinase (betaARK) and GRK6 expression and activity in myelomonocytic and lymphoid cells. In the promyelocytic cell line HL-60, GRK6 protein levels and activity rose twofold to fourfold over the course of treatment with agents that induce differentiation toward either the myeloid (dimethyl sulfoxide and retinoic acid) or monocytic [1alpha,25(OH)2-vitamin D3] lineage, whereas betaARK protein levels and activity were only slightly altered. In contrast, treatment with phorbol 12,13-myristic acetate (PMA) led to a reduction in steady-state levels and activity of both betaARK and GRK6. Treatment of human lymphocytes with several polyclonal activators (phytohemagglutinin, anti-CD3 antibody and interleukin-2) resulted in enhanced betaARK and GRK6 mRNA and protein levels and increased activity of both kinases. In contrast, PMA and calcium ionophore treatment significantly elevated GRK6 protein levels, while decreasing betaARK expression. These data demonstrate that betaARK and GRK6 expression are differentially regulated during myelomonocytic cell development and lymphocyte activation.

Cyclic AMP-Dependent Protein Kinases↗
PubMed1996

Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor.

The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.

Animals↗
PubMed1996

G protein-coupled receptor kinase GRK2 is a phospholipid-dependent enzyme that can be conditionally activated by G protein betagamma subunits.

G protein-coupled receptor kinases (GRKs) mediate agonist-dependent phosphorylation of G protein-coupled receptors (GPRs) and initiate homologous receptor desensitization. Previously, we reported that charged phospholipids directly interacted with the two GRK isoforms, GRK2 and GKR3, via a pleckstrin homology (PH) domain to regulate GRK activity (DebBurman, S. K., Ptasienski, J., Boetticher, E., Lomasney, J. W., Benovic, J. L., and Hosey, M. M. (1995) J. Biol. Chem. 270: 5742-5747). Here, evidence is provided to support the hypothesis that charged phospholipids are required for agonist-dependent phosphorylation of receptors by GRK2. In the absence of charged phospholipids, the purified human m2 muscarinic acetylcholine receptor (hm2mAChR) reconstituted in pure phosphatidylcholine vesicles or in a noninhibitory detergent was not a substrate for GRK2. However, these receptor preparations were stoichiometrically phosphorylated in an agonist-dependent manner upon addition of charged phospholipids. The known ability of G protein betagamma subunits to stimulate mAChR phosphorylation also was found to be absolutely dependent on the presence of charged phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP2). Phospholipids also regulated GRK-mediated phosphorylation of casein, a nonreceptor-soluble substrate. Among lipids tested, lipid inositol phosphates, PIP2 and phosphatidylinositol 4-monophosphate, were found to be the most potent activators of GRK2 and were the only lipids that regulated GRK2 in a complex biphasic manner. At low micro concentrations, PIP2 activated GRK2 via an interaction with the GRK pleckstrin homology domain; however, at high micro concentrations, PIP2 inhibited GRK2, apparently via another mechanism. PIP2-mediated inhibition could be partly relieved by increasing ATP. The results support the hypothesis that GRK2 is a lipid-dependent protein kinase that requires charged phospholipids for enzyme activation, for regulation by Gbetagamma subunits, and potentially for membrane association.

Adenosine Triphosphate↗
PubMed1996

Molecular basis for subtype-specific desensitization of inhibitory adenosine receptors. Analysis of a chimeric A1-A3 adenosine receptor.

The differing effects of short-term agonist exposure on the two inhibitory adenosine receptor (AR) subtypes have been examined using Chinese hamster ovary cells stably expressing the hemagglutinin epitope-tagged human A1AR and rat A3AR. Under conditions in which exposure of transfected cells to 5 microM (-)-(R)-N6-(phenylisopropyl)adenosine resulted in the functional desensitization and phosphorylation of the A3AR, neither property was exhibited by the A1AR. However, a stably expressed chimeric A1-A3AR, termed A1CT3AR, in which the C-terminal domain of the A1AR distal to its predicted palmitoylation site was replaced by the corresponding region of the A3AR, was able to undergo functional desensitization and agonist-stimulated phosphorylation in a manner similar to that exhibited by the A3AR. Moreover, purified G-protein-coupled receptor kinases 2, 3, and 5 were each capable of enhancing the agonist-dependent phosphorylation of the A3AR and A1CT3AR in vitro. Taken together, these data demonstrate that the C-terminal domain of the A3AR distal to its predicted palmitoylation site is responsible for this receptor's ability to undergo a rapid agonist-dependent desensitization and are consistent with a model in which phosphorylation of the A3AR within this domain by one or more G-protein-coupled receptor kinases initiates the desensitization process.

Adenosine↗
PubMed1996

Effect of different G protein-coupled receptor kinases on phosphorylation and desensitization of the alpha1B-adrenergic receptor.

The alpha1B-adrenergic receptor (alpha1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of beta-adrenergic receptor kinase (betaARK) 1 (GRK2) or 2 (GRK3) could increase epinephrine-induced phosphorylation of the wild type alpha1BAR above basal as compared to that of the receptor expressed alone. On the other hand, overexpression of the dominant negative betaARK (K220R) mutant impaired agonist-induced phosphorylation of the receptor. Overexpression of GRK6 could also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhanced basal but not agonist-induced phosphorylation of the alpha1BAR. Increasing coexpression of betaARK1 or betaARK2 resulted in the progressive attenuation of the alpha1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of betaARK1 or 2 at low levels did not significantly impair the PI response mediated by the truncated alpha1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response was also observed for the wild type alpha1BAR, but not for its truncated mutant, when the receptor was coexpressed with beta-arrestin 1 or beta-arrestin 2. Despite their pronounced effect on phosphorylation of the alpha1BAR, overexpression of GRK5 or GRK6 did not affect the receptor-mediated response. In conclusion, our results provide the first evidence that betaARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the alpha1BAR. They also identify the alpha1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the alpha1BAR remain to be elucidated.

Adrenergic beta-Agonists↗
PubMed1996

High affinity binding of beta-adrenergic receptor kinase to microsomal membranes. Modulation of the activity of bound kinase by heterotrimeric G protein activation.

The beta-adrenergic receptor kinase (beta ARK) modulates beta-adrenergic and other G protein-coupled receptors by rapidly phosphorylating agonist-occupied receptors at the plasma membrane. We have recently shown that beta ARK also associates with intracellular microsomal membranes both "in vitro" and "in situ" (García-Higuera, I., Penela, P., Murga, C., Egea, G., Bonay, P., Benovic, J. L., and Mayor, F., Jr. (1994) J. Biol. Chem. 269, 1348-1355), thus suggesting a complex modulation of the subcellular distribution of beta ARK. In this report, we used recombinant [35S]methionine-labeled beta ARK to show that this kinase interacts rapidly with a high affinity binding site (Kd of 20 +/- 1 nM) present in salt-stripped rat liver microsomal membranes. Although beta ARK binding is not modulated by membrane preincubation with G protein activators, the activity of bound beta ARK toward rhodopsin or a synthetic peptide substrate was markedly enhanced upon stimulation of the endogenous heterotrimeric G proteins present in the microsomal membranes by AIF4- or mastoparan/guanosine 5'-(3-O-thio)triphosphate, thus strongly suggesting a functional link between these proteins and membrane-associated beta ARK. Interestingly, beta ARK association with microsomal membranes is not significantly affected by a fusion protein derived from the carboxyl terminus of beta ARK1 (the proposed location of the beta gamma subunit binding site), whereas it is markedly inhibited by fusion proteins corresponding to the amino-terminal region of the kinase. The main determinants of binding appear to be localized to an approximately 60-amino acid residue stretch (residues 88 to 145). Our results further indicate a functional relationship between beta ARK and heterotrimeric G proteins in different intracellular organelles, and suggest that additional proteins may be involved in modulating the cellular localization of the kinase through a new targeting domain of beta ARK.

Animals↗
PubMed1996

Effects of inflammation and acute beta-agonist inhalation on beta 2-AR signaling in human airways.

Although alterations in beta 2-adrenergic receptor (AR) responsiveness may in part explain reports linking deterioration of asthma control with beta-agonist treatment of asthmatics, few data exist on beta 2-AR regulation in human airway cells. We have employed a bronchoscopy model to examine inflammation- and beta-agonist-induced alterations in human bronchial epithelial cell beta 2-AR density and responsiveness. Allergic asthmatic subjects participated in 2-day protocols examining airways before and 24 h after segmental antigen challenge (SAC) with ragweed. To assess the effect of acute beta-agonist exposure, bronchoscopies were performed both with (+ beta-Ag) and without (-beta-Ag) inhalation of beta-agonist 30 min before the procedure. Measurements of inflammatory cell infiltration were obtained by analysis of bronchoalveolar lavage fluid, and beta 2-AR density and responsiveness were examined in bronchial epithelial cells obtained by bronchoscopic brushing. Neither SAC nor acute beta-agonist administration alone significantly affected epithelial cell beta 2-AR density. beta-Agonist-stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) generation was significantly lower in the + beta-Ag groups compared with the-beta-Ag group, demonstrating acute agonist-specific beta 2-AR desensitization in vivo. SAC caused a small, statistically insignificant reduction in beta-Agonist-stimulated cAMP production in both -beta-Ag or + beta-Ag groups. These lata suggest that acute beta-agonist inhalation, but not airway inflammation, significantly reduces maximal beta 2-AR responsiveness in airway cells.

Albuterol↗
PubMed1995

Agonist-dependent phosphorylation and desensitization of the rat A3 adenosine receptor. Evidence for a G-protein-coupled receptor kinase-mediated mechanism.

A3 adenosine receptor (A3AR) activation contributes to both the cardioprotective and antihypertensive effects of adenosine. To date, no studies have examined the mechanisms by which this receptor undergoes rapid homologous desensitization. Therefore, a functional hemagglutinin epitope-tagged A3AR has been stably expressed in Chinese hamster ovary cells, and its regulation by the AR agonist 5'-N-ethylcarboxamidoadenosine (NECA) has been studied. Cellular exposure to NECA induces rapid (t1/2 = approximately 1 min) A3AR phosphorylation on serine and threonine residues. This is associated with a functional desensitization and a 30-40% reduction in the number of high affinity agonist binding sites as determined by radioligand binding assays. Activation of second messenger-regulated kinases could not mimic the effect of NECA, suggesting a role for G-protein-coupled receptor kinases (GRKs). In vitro phosphorylation assays demonstrate that phosphorylation of agonist-occupied A3ARs is enhanced by GRK2 and that cellular pretreatment with NECA dramatically inhibits subsequent GRK2-mediated phosphorylation in vitro. Therefore, the A3AR is phosphorylated in situ by a kinase similar or identical to GRK2, and this may be involved in rapid functional desensitization of the A3AR.

Adenosine↗
PubMed1995

Desensitization and internalization of the m2 muscarinic acetylcholine receptor are directed by independent mechanisms.

The phenomenon of acute desensitization of G-protein-coupled receptors has been associated with several events, including receptor phosphorylation, loss of high affinity agonist binding, receptor:G-protein uncoupling, and receptor internalization. However, the biochemical events underlying these processes are not fully understood, and their contributions to the loss of signaling remain correlative. In addition, the nature of the kinases and the receptor domains which are involved in modulation of activity have only begun to be investigated. In order to directly measure the role of G-protein-coupled receptor kinases (GRKs) in the desensitization of the m2 muscarinic acetylcholine receptor (m2 mAChR), a dominant-negative allele of GRK2 was used to inhibit receptor phosphorylation by endogenous GRK activity in a human embryonic kidney cell line. The dominant-negative GRK2K220R reduced agonist-dependent phosphorylation of the m2 mAChR by approximately 50% and prevented acute desensitization of the receptor as measured by the ability of the m2 mAChR to attenuate adenylyl cyclase activity. In contrast, the agonist-induced internalization of the m2 mAChR was unaffected by the GRK2K220R construct. Further evidence linking receptor phosphorylation to acute receptor desensitization was obtained when two deletions of the third intracellular loop were made which created m2 mAChRs that did not become phosphorylated in an agonist-dependent manner and did not desensitize. However, the mutant mAChRs retained the ability to internalize. These data provide the first direct evidence that GRK-mediated receptor phosphorylation is necessary for m2 mAChR desensitization; the likely sites of in vivo phosphorylation are in the central portion of the third intracellular loop (amino acids 282-323). These results also indicate that internalization of the m2 receptor is not a key event in desensitization and is mediated by mechanisms distinct from GRK phosphorylation of the receptor.

Alleles↗
PubMed1995

The beta-adrenergic receptor kinase (GRK2) is regulated by phospholipids.

The beta-adrenergic receptor kinase (beta ARK) is a member of growing family of G protein coupled receptor kinases (GRKs). beta ARK and other members of the GRK family play a role in the mechanism of agonist-specific desensitization by virtue of their ability to phosphorylate G protein-coupled receptors in an agonist-dependent manner. beta ARK activation is known to occur following the interaction of the kinase with the agonist-occupied form of the receptor substrate and heterotrimeric G protein beta gamma subunits. Recently, lipid regulation of GRK2, GRK3, and GRK5 have also been described. Using a mixed micelle assay, GRK2 (beta ARK1) was found to require phospholipid in order to phosphorylate the beta 2-adrenergic receptor. As determined with a nonreceptor peptide substrate of beta ARK, catalytic activity of the kinase increased in the presence of phospholipid without a change in the Km for the peptide. Data obtained with the heterobifunctional cross-linking agent N-3-[125I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl)-c ysteine ([125I]ACTP) suggests that the activation by phospholipid was associated with a conformational change in the kinase. [125I]ACTP incorporation increased 2-fold in the presence of crude phosphatidylcholine, and this increase in [125I]ACTP labeling is completely blocked by the addition of MgATP. Furthermore, proteolytic mapping was consistent with the modification of a distinct site when GRK2 was labeled in the presence of phospholipid. While an acidic phospholipid specificity was demonstrated using the mixed micelle phosphorylation assay, a notable exception was observed with PIP2. In the presence of PIP2, kinase activity as well as [125I]ACTP labeling was inhibited. These data demonstrate the direct regulation of GRK2 activity by phospholipids and supports the hypothesis that this effect is the result of a conformational change within the kinase.

Amino Acid Sequence↗