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J L Benovic

Publications and source records attributed to J L Benovic.

At least 109 records · Page 6Linked to original sources
PubMed1995

Modulation by GM1 ganglioside of beta 1-adrenergic receptor-induced cyclic AMP formation in Sf9 cells.

The effect of ganglioside GM1 on isoproterenol-induced cAMP accumulation was studied in insect Sf9 cells expressing the human beta 1-adrenergic receptor by infection with recombinant baculovirus. When such Sf9 cells were treated with isoproterenol plus IBMX, intracellular cAMP formation increased approximately 10-fold over the basal level. Preincubation of the baculovirus-infected cells with GM1 for 1 h caused a concentration-dependent inhibition of the isoproterenol-induced cAMP accumulation. Phosphatidylserine, GM3, GT1b and a bovine brain ganglioside preparation lacking GM1 did not cause significant inhibition. Forskolin-induced cAMP accumulation was not affected by the GM1 treatment. Inhibition of isoproterenol-induced cAMP formation by GM1 was not observed in Sf9 cells expressing beta 2-adrenergic receptor instead of the beta 1-adrenergic receptor. Binding studies with (-)-[3H]CGP12177 showed that preincubation with GM1 significantly reduced the affinity of antagonist binding to the beta 1-adrenergic receptor. These results suggest that GM1 or related ganglioside structure(s) may function as natural modulator(s) of the beta 1-adrenergic receptor.

Adenosine Monophosphate↗
PubMed1995

Sequestration of a G-protein beta gamma subunit or ADP-ribosylation of Rho can inhibit thrombin-induced activation of platelet phosphoinositide 3-kinases.

Stimulation of platelets by thrombin leads to an increased association of activated phosphoinositide 3-kinase (PI 3-K) with a membrane cytoskeletal fraction (CSK). Activation of PI 3-K is dependent upon GTP-binding protein(s), since PI 3-K in permeabilized platelets is stimulated by GTP gamma S (guanosine 5'-3-O-(thio)triphosphate), and stimulation of platelet cytosolic PI 3-K by GTP gamma S requires a functional small G-protein, Rho. Recent reports indicate that cytosolic PI 3-Ks can also be activated by the beta gamma subunits of heterotrimeric G-proteins (G beta gamma). We now report that the activated PI 3-K that is associated with CSK can be inhibited by a recombinant protein containing the G beta gamma-binding pleckstrin homology domain of beta-adrenergic receptor kinase 1 (beta ARK-PH). Inhibition is blocked by G beta gamma. PI 3-K in nonactivated platelet CSK is activated by GTP gamma S but unaffected by beta ARK-PH or G beta gamma. Western blots indicate that activated platelet CSK contains a novel 110-kDa PI 3-K(gamma) that has been shown to be stimulated by G beta gamma and to lack binding sites for the 85-kDa subunit of conventional PI 3-K. PI 3-K in immunoprecipitates obtained via p85 subunit-directed antibodies can be activated by GTP gamma S but not by G beta gamma. PI 3-K that is stimulatable by G beta gamma remains soluble, as does PI 3-K(gamma), and is unaffected by Rho. In contrast, ADP-ribosylation of Rho present in p85 immunoprecipitates is inhibitory. Further, activation of PI 3-K in permeabilized platelets exposed to thrombin or GTP gamma S is inhibited by beta ARK-PH and/or Rho-specific ADP-ribosylating enzymes. We conclude that Rho and G beta gamma each, respectively, contributes to the activation of different PI 3-Ks (p85-containing heterodimer and PI 3-K (gamma)) in thrombin-stimulated platelets.

Adenosine Diphosphate Ribose↗
PubMed1995

Lipid-mediated regulation of G protein-coupled receptor kinases 2 and 3.

G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.

Animals↗
PubMed1995

Visual arrestin binding to rhodopsin. Diverse functional roles of positively charged residues within the phosphorylation-recognition region of arrestin.

Arrestin plays a critical role in quenching phototransduction via its ability to specifically interact with the phosphorylated light-activated form of the visual receptor rhodopsin. In an effort to identify the residues involved in interaction with the phosphorylated C terminus of rhodopsin, we introduced point mutations into a basic region in visual arrestin previously implicated in phosphorylation-recognition (residues 163-189). A total of nine point mutations were made, each substituting a neutral hydrophilic residue for a positively charged Lys, Arg, or His. The functional consequences of these mutations were then analyzed by comparing the binding of full-length and truncated wild-type and mutant arrestin to various functional forms of rhodopsin. These studies demonstrate that Arg-171, Arg-175, and Lys-176 in bovine arrestin play a primary role in phosphate interaction, while Lys-166 and Lys-167 likely play a minor role in phosphate binding. In contrast, Lys-163 and His-179 appear to play a regulatory role, while Arg-182 and Arg-189 are not directly involved in arrestin binding to rhodopsin. Arg-175 also appears to function as a phosphorylation-sensitive trigger since charge neutralization by mutagenesis enables arrestin-R175N to bind to light-activated rhodopsin as well as wild-type arrestin binds to phosphorylated light-activated rhodopsin. The implications of these findings for the sequential multisite binding of arrestin to rhodopsin are discussed.

Acetates↗
PubMed1995

Phosphorylation of the N-formyl peptide receptor carboxyl terminus by the G protein-coupled receptor kinase, GRK2.

Attenuation of receptor-mediated signal amplification in response to external stimuli, an essential step in the balance of cellular activation, may be mediated by receptor phosphorylation. We have recently shown that the carboxyl-terminal cytoplasmic domain of the N-formyl peptide receptor (FPR) interacts with G proteins and demonstrate here that this same region of the FPR is specifically phosphorylated by a neutrophil cytosolic kinase with properties similar to the G protein-coupled receptor kinase, GRK2. Both kinase activities show a lack of sensitivity toward protein kinase A, protein kinase C, and tyrosine kinase inhibitors but demonstrate almost identical sensitivity toward the kinase inhibitor heparin. Kinetic studies demonstrated that GRK2 has a Km for the carboxyl-terminal domain of the FPR of approximately 1.5 microM and that denaturation of the substrate results in an almost complete loss of phosphorylation. Comparative studies reveal that GRK3 has approximately 50% of the activity of GRK2 toward the FPR carboxyl terminus, whereas GRK5 and GRK6 have no detectable activity. Site-directed mutagenesis of numerous regions of the FPR carboxyl terminus demonstrated that, whereas Glu326/Asp327 and Asp333 are critical for phosphorylation, the carboxyl-terminal 10 amino acids are not required. Simultaneous substitution of Thr334, Thr336, Ser338, and Thr339 resulted in an approximately 50% reduction in phosphorylation, whereas simultaneous substitution of the upstream Ser328, Thr329, Thr331, and Ser332 or merely the Ser328 and Thr329 residues resulted in an approximately 80% reduction in phosphorylation. The introduction of negatively charged glutamate residues for Ser328 and Thr329 or Thr331 and Ser332 resulted in marked stimulation of phosphorylation. These results suggest a hierarchical mechanism in which phosphorylation of amino-terminal serine and threonine residues is required for the subsequent phosphorylation of carboxyl-terminal residues. These results provide the first direct evidence that an intracellular domain of a chemoattractant receptor is a high affinity substrate for GRK2 and further suggest a role for GRK2 or a closely related kinase in the attenuation of receptor-mediated activation of inflammatory cells.

Amino Acid Sequence↗
PubMed1995

Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, beta 2-adrenergic, and m2 muscarinic cholinergic receptors.

Arrestins play an important role in quenching signal transduction initiated by G protein-coupled receptors. To explore the specificity of arrestin-receptor interaction, we have characterized the ability of various wild-type arrestins to bind to rhodopsin, the beta 2-adrenergic receptor (beta 2AR), and the m2 muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found to be the most selective arrestin since it discriminated best between the three different receptors tested (highest binding to rhodopsin) as well as between the phosphorylation and activation state of the receptor (> 10-fold higher binding to the phosphorylated light-activated form of rhodopsin compared to any other form of rhodopsin). While beta-arrestin and arrestin 3 were also found to preferentially bind to the phosphorylated activated form of a given receptor, they only modestly discriminated among the three receptors tested. To explore the structural characteristics important in arrestin function, we constructed a series of truncated and chimeric arrestins. Analysis of the binding characteristics of the various mutant arrestins suggests a common molecular mechanism involved in determining receptor binding selectivity. Structural elements that contribute to arrestin binding include: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of a receptor, without directly participating in receptor interaction; 2) a basic N-terminal domain that directly participates in receptor interaction and appears to serve a regulatory role via intramolecular interaction with the C-terminal acidic region; and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity. A comparative structure-function model of all arrestins and a kinetic model of beta-arrestin and arrestin 3 interaction with receptors are proposed.

Animals↗
PubMed1995

Multiple mechanisms involving protein phosphorylation are linked to desensitization of muscarinic receptors.

Agonists induce phosphorylation of m2 muscarinic receptors (mAChR) in several cell types. This phosphorylation correlates with desensitization. The mechanisms underlying mAChR phosphorylation have been investigated using several in vitro approaches. Protein kinase C phosphorylated the purified and reconstituted m2 mAChR to a stoichiometry of approximately 5 mols P/mol receptor; this phosphorylation resulted in the decreased ability of receptors to activate G-proteins. Although the phosphorylation by PKC was not modulated by agonist binding to the mAChR, heterotrimeric G-proteins were able to completely block the PKC-mediated effects. If significant receptor/G-protein coupling occurs in vivo, agonists would be required to promote dissociation of the G-proteins from the receptors and reveal the phosphorylation sites for PKC. Members of the G-protein coupled receptor kinase (GRK) family also phosphorylated the purified and reconstituted m2 mAChR. In contrast to PKC, the GRKs phosphorylated the m2 mAChR strictly in an agonist-dependent manner. GRK mediated phosphorylation perturbed receptor/G-protein coupling. In addition, phosphorylation allowed for arrestin binding to the m2 mAChR which should further contribute to desensitization. Using a new strategy that does not require purification and reconstitution of receptors for GRK studies, the m3 mAChR were revealed as substrates for the GRKs. For both the m2 and m3 receptor subtypes, the most effective kinases were GRK 2 and 3. Phosphorylation of the receptors by these enzymes was stimulated by low concentrations of G-proteins and by membrane phospholipids. Thus, multiple mechanisms involving protein phosphorylation appear to contribute to the overall process of mAChR desensitization.

Animals↗
PubMed1995

Expression and function of the beta-adrenergic receptor coupled-adenylyl cyclase system on human airway epithelial cells.

Beta-adrenergic agonist-mediated activation of the beta receptor coupled-adenylyl cyclase (beta AR-AC) system expressed by human airway epithelial cells alters airway function. However, little is known about the magnitude of expression, subtype, and function of the beta receptor-adenylyl cyclase (beta AR-AC) system in human airway epithelial cells from healthy, nonsmoking subjects. Therefore, we characterized beta AR number and subtype and the cAMP response to isoproterenol (iso) in acutely dissociated human tracheocytes harvested from 22 healthy, nonsmoking adults during fibroptic bronchoscopy. Moreover, because the regulation of beta AR-AC system function in response to beta-agonists or inflammatory mediators released into the airway in asthma is poorly understood, we examined the cAMP response to iso after 30 min exposure of cells to iso or the protein kinase C activator, sn-1,2-dioctanoyl glycerol (diC8). The beta AR-AC system was highly expressed and functional in human airway epithelial cells. Group mean beta AR density (i.e., Bmax), equilibrium dissociation constant (Kd), and the percentage of beta 2AR subtypes assessed by radioligand binding were approximately 8,900 receptors/cell, 45 pM, and approximately 80%, respectively. Mean maximum cAMP production was approximately 42 pmol/10(5) cells, and the mean EC50 of the response to iso was 131 nM. However, Bmax and cAMP responses to iso varied considerably across subjects. For example, Bmax varied ninefold, and the EC50 of the cAMP response varied 39-fold interindividually. The EC50 was inversely related to beta AR density (r = -0.81, p < 0.05), suggesting that sensitivity of the cAMP response to iso was in part dependent on beta AR density. In all experiments, cAMP responses to iso stimulation were markedly desensitized in dose-dependent fashion by 30 min pretreatment with iso or diC8. For example, pretreatment with iso 10 microM or diC8 100 microM reduced maximum cAMP production to 22 and 63% of control values, respectively. These data indicate that: (1) the beta AR-AC system is highly expressed on acutely dissociated airway epithelial cells from normal adult, but beta AR expression and its functional coupling to adenylyl cyclase vary considerably interindividually; and (2) the beta AR-AC system of normal human airway epithelial cells is rapidly desensitized by exposure to beta-adrenergic agonists or activators of PKC.

Adenylyl Cyclases↗
PubMed1995

Agonist-dependent phosphorylation of human muscarinic receptors in Spodoptera frugiperda insect cell membranes by G protein-coupled receptor kinases.

Agonist-dependent phosphorylation of G protein-coupled receptors (GPRs) by G protein-coupled receptor kinases (GRKs) is proposed to be a key event initiating homologous receptor desensitization. A technical limitation hindering identification of GPRs as GRK substrates has been the necessity to use purified and reconstituted receptors in GRK assays. Here, the human m2 and human m3 (hm3) muscarinic cholinergic receptors (mAChRs), which couple to attenuation of adenylyl cyclase and stimulation of phospholipase C, respectively, were expressed in Spodoptera frugiperda insect cells and an in vitro approach to studying GPR phosphorylation by GRKs in crude membranes was developed. The m2 mAChR, a known substrate of certain GRKs, was used to validate the approach. The GRK isoform beta-adrenergic receptor kinase (beta ARK)1 phosphorylated the membrane-bound human m2 mAChRs in an agonist-dependent manner. The results demonstrated that endogenous membrane-bound beta gamma subunits of G proteins stimulated the phosphorylation of the membrane-bound m2 mAChR. To reveal new GRK substrates, we tested the expressed hm3 mAChRs. The membrane-bound hm3 mAChRs were phosphorylated by beta ARK1 in an agonist-dependent, G beta gamma-enhanced manner. This is the first demonstration that hm3 mAChRs can serve as substrates for GRKs. The stoichiometry of receptor phosphorylation was approximately 2 mol of phosphate/mol of receptors in the absence of G beta gamma and approximately 4 mol of phosphate/mol of receptors upon addition of G beta gamma. When the specificity of various GRKs towards mAChRs was assessed, beta ARK2 phosphorylated the agonist-activated hm3 mAChRs as efficiently as did beta ARK1; however, neither GRK5 nor GRK6 significantly phosphorylated the hm3 mAChRs under similar conditions. The approach of studying GRK-mediated phosphorylation of GPRs in their membrane-bound state identified the hm3 mAChRs as new substrates for GRKs. This approach should be valuable in identifying other new substrates of GRKs and should aid in studies that elucidate GRK/GPR pairing.

Animals↗
PubMed1994

Expression, purification, and characterization of the G protein-coupled receptor kinase GRK6.

G protein-coupled receptor kinases (GRKs), such as rhodopsin kinase and beta-adrenergic receptor kinase (beta ARK), are involved in mediating agonist-specific phosphorylation and desensitization of G protein-coupled receptors. GRK6 is the most recently identified member of the GRK family and displays higher homology with GRK5 (70.1% amino acid identity) and IT11 (68.5%) compared to beta ARK (37.4%) and rhodopsin kinase (47.1%). To further characterize GRK6, it has been overexpressed in Sf9 cells and purified to homogeneity by sequential chromatography on SP-Sepharose and heparin-Sepharose columns. GRK6 shares a number of in vitro characteristics with GRK5, including potent inhibition by heparin and dextran sulfate (IC50 values of approximately 15 and approximately 7 nM, respectively), hyperstimulation by polycations, and preference for phosphorylation of non-acidic peptides. Rhodopsin and the beta 2-adrenergic and m2 muscarinic cholinergic receptors serve as stimulus-dependent substrates for GRK6, but with stoichiometries significantly lower than achieved by GRK5 and beta ARK. Additionally, GRK6 does not undergo significant autophosphorylation even though it contains residues identical to those that are autophosphorylated in GRK5 and rhodopsin kinase. These data extend our knowledge of a growing family of receptor-specific kinases and suggest that GRK6 has a substrate specificity distinct from beta ARK, rhodopsin kinase, and GRK5.

Amino Acid Sequence↗
PubMed1994

Structure of the human gene encoding the beta-adrenergic receptor kinase.

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied forms of the beta 2-adrenergic receptor and related G protein-coupled receptors. beta ARK is one of the best characterized members of a growing family of G protein-coupled receptor kinases. In this article we report the isolation and structural organization of the human beta ARK gene. The gene spans approximately 23 kilobases and is composed of 21 exons interrupted by 20 introns. Exon sizes range from 52 bases (exon 7) to over 1200 bases (exon 21), intron sizes from 68 bases (intron L) to 10.8 kilobases (intron A). The splice sites for donor and acceptor were in agreement with the canonical GT/AG rule. Functional regions of beta ARK are described with respect to their location within the exon-intron organization of the gene. Primer extension and RNase protection assays suggest a major transcription start site approximately 246 bases upstream of the start ATG. Sequence analysis of the 5'-flanking/promoter region reveals many features characteristic of mammalian housekeeping genes, i.e. the lack of a TATA box, an absent or nonstandard positioned CAAT box, high GC content, and the presence of Sp1-binding sites. The extraordinarily high GC content of the 5'-flanking region (> 80%) helps define this region as a CpG island that may be a principal regulator of beta ARK expression.

Amino Acid Sequence↗
PubMed1994

A beta-adrenergic receptor kinase dominant negative mutant attenuates desensitization of the beta 2-adrenergic receptor.

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the activated form of the beta 2-adrenergic receptor (beta 2AR) and related G protein-coupled receptors. To further elucidate the role of beta ARK in receptor desensitization, we generated a beta ARK dominant negative mutant by converting an invariant lysine residue in the protein kinase catalytic domain to an arginine. Expressed and purified beta ARK-K220R was able to inhibit wild type beta ARK phosphorylation of the beta 2AR in vitro. When stably transfected into human bronchial epithelial BEAS-2B cells, beta ARK-K220R promoted a > 2-fold increase in beta-agonist-stimulated cAMP production without affecting beta 2AR sequestration. In contrast, beta ARK-K220R had no effect on the desensitization of the prostaglandin E2 receptor response in BEAS-2B cells. These findings directly demonstrate a role for beta ARK in desensitization of the beta 2AR in intact cells and establish the potential utility of using dominant negative mutants to elucidate the substrate specificity of G protein-coupled receptor kinases.

Animals↗
PubMed1994

Phospholipid-stimulated autophosphorylation activates the G protein-coupled receptor kinase GRK5.

G protein-coupled receptor kinases (GRKs) play an important role in mediating agonist-specific desensitization of numerous G protein-coupled receptors. GRK5, a recently identified member of the GRK family, undergoes a rapid phospholipid-stimulated autophosphorylation to a stoichiometry of approximately 2 mol of phosphate/mol of GRK5. The ability of phospholipids to stimulate autophosphorylation is largely blocked by a glutathione S-transferase fusion protein containing the last 102 amino acids of GRK5 (amino acids 489-590), suggesting that this is a primary region involved in GRK5/phospholipid interaction. Phosphoamino acid determination and mutagenesis studies demonstrate that autophosphorylation of GRK5 occurs primarily at residues Ser-484 and Thr-485. Expression and characterization of a mutant GRK5 that does not autophosphorylate (S484A and T485A) reveals that the mutant has a approximately 15-20-fold reduced ability to phosphorylate the beta 2-adrenergic receptor and rhodopsin compared to wild type GRK5. These results suggest that phospholipid-stimulated autophosphorylation may represent a novel mechanism for membrane association and regulation of GRK5 activity.

Amino Acid Sequence↗
PubMed1994

Visual arrestin binding to rhodopsin. Intramolecular interaction between the basic N terminus and acidic C terminus of arrestin may regulate binding selectivity.

Visual arrestin plays an important role in quenching phototransduction via its ability to preferentially bind to phosphorylated light-activated rhodopsin (P-Rh*). Recently we proposed a mechanism for the binding of visual arrestin to P-Rh* that helps to explain the nature of the conformational changes in arrestin observed upon binding. This mechanism involves a multisite interaction between arrestin and P-Rh* and implies an interaction between the C-terminal and N-terminal domains of arrestin. To obtain further insight into the mechanism of arrestin-rhodopsin interaction we have characterized the ability of polyanions to inhibit the interaction of wild type and mutant arrestins to different functional forms of rhodopsin. These studies reveal that: 1) heparin is most potent at inhibiting arrestin binding to dark phosphorylated rhodopsin > light-activated rhodopsin > P-Rh*; 2) C-terminal deletions in arrestin increase arrestin sensitivity to heparin inhibition while an N-terminal deletion (residues 2-16) decreases heparin inhibition; 3) the sensitivity of chimeric arrestins to heparin inhibition is determined by the origin of the N terminus of the chimera; and 4) heparin also inhibits arrestin binding to truncated 329G-Rh*, suggesting it does not mimic the phosphorylated C terminus of rhodopsin. Taken together, these data suggest that heparin mimics the regulatory acidic C terminus of arrestin. Since the basic N-terminal region of arrestin appears to serve as a site of heparin binding it is a likely candidate to be involved in the intramolecular interaction with the C-terminal region. The interaction of the N- and C-terminal domains of arrestin may control the conformational rearrangements in arrestin that occur upon binding to P-Rh*.

Animals↗
PubMed1994

Arrestin-rhodopsin interaction. Multi-site binding delineated by peptide inhibition.

Visual arrestin modulates the intracellular response of retinal rod cells to light by specifically binding to the phosphorylated light-activated form of the photoreceptor rhodopsin (P-Rh*). In order to characterize the molecular interaction between rhodopsin and arrestin, we have studied the ability of synthetic peptides from the proposed cytoplasmic loops of rhodopsin to inhibit arrestin binding. A third cytoplasmic loop peptide competed most effectively for arrestin binding to P-Rh*, exhibiting an IC50 of 34 microM, while a first cytoplasmic loop peptide weakly inhibited binding with an IC50 of approximately 1100 microM. The first and third cytoplasmic loop peptides also inhibited P-Rh* interaction with both ARR[delta (2-16)-404], an arrestin mutant that lacks residues 2-16, and ARR[1-191], a mutant that contains only the amino half of arrestin. However, the third loop peptide had an approximately 5-fold lower affinity at inhibiting the binding of ARR[1-191] to P-Rh*. While the first and third loop peptides also inhibited arrestin binding to light-activated rhodopsin and a truncated rhodopsin lacking its C-terminal sites of phosphorylation, the peptides modestly enhanced arrestin binding to phosphorylated dark rhodopsin. These results suggest that the third and, to a lesser extent, the first cytoplasmic loops of rhodopsin may play an important role in arrestin binding to light-activated forms of rhodopsin.

Amino Acid Sequence↗
PubMed1994

Expression, purification, and characterization of the G protein-coupled receptor kinase GRK5.

G protein-coupled receptor kinases (GRKs) such as rhodopsin kinase and the beta-adrenergic receptor kinase (beta ARK) play an important role in agonist-specific phosphorylation and desensitization of G protein-coupled receptors. GRK5 is a recently identified member of the GRK family that has greater homology with rhodopsin kinase than with beta ARK. To further characterize the activity of GRK5, it has been overexpressed in Sf9 insect cells and purified by successive chromatography on S-Sepharose and Mono S columns. GRK5 phosphorylates the beta 2-adrenergic receptor (beta 2AR), m2 muscarinic cholinergic receptor, and rhodopsin in an agonist-dependent manner to maximal stoichiometries of approximately 2.5, 1.5, and 1 mol of phosphate/mol of receptor, respectively, with Km values of approximately 0.5 microM for the beta 2AR, approximately 16 microM for rhodopsin, and approximately 24 microM for ATP. Peptide phosphorylation studies suggest that in contrast to beta ARK and rhodopsin kinase, GRK5 preferentially phosphorylates on nonacidic peptides with a Km of approximately 1.5 mM. Heparin and dextran sulfate were found to be potent inhibitors of GRK5 with IC50 values of approximately 1 nM, thereby being at least 150-fold more potent on GRK5 than on beta ARK. GRK5 can also be activated by polycations, with 10 microM polylysine promoting an approximately 2.6-fold activation. Overall, these studies demonstrate that GRK5 has unique properties that distinguish it from other members of the GRK family and that likely play an important role in modulating its mechanism of action.

Amino Acid Sequence↗
PubMed1994

Association of the regulatory beta-adrenergic receptor kinase with rat liver microsomal membranes.

beta-Adrenergic receptor kinase (beta ARK) is a regulatory enzyme involved in the modulation of beta-adrenergic and other G protein-coupled receptors. It has been described that beta ARK is a cytosolic protein that transiently translocates to the plasma membrane in order to specifically phosphorylate agonist-occupied receptors. In this report, we used beta ARK-specific antibodies to demonstrate that a significant amount of this kinase is present in rat liver microsomal membranes. beta ARK seems to be peripherally associated with the cytosolic side of microsomal membranes since it can be stripped from the membranes by mild salt treatment. Cell-free association experiments indicate that the interaction of beta ARK is reversible, saturable, and strongly inhibited by protease or heat treatment of the microsomes, thus suggesting that beta ARK interacts with a protein component of the microsomal membrane. Gradient fractionation studies indicate that the highest beta ARK-specific activity co-migrates with endoplasmic reticulum enzymatic markers. Furthermore, indirect immunofluorescence and immunogold electron microscopy experiments performed in cultured cells using affinity-purified anti-beta ARK antibodies are consistent with this subcellular localization pattern. Taken together, our data suggest that several beta ARK pools (i.e. microsome-bound, plasma membrane-bound, and cytosolic) may exist inside the cell. Such results are in line with recent reports showing that proteins involved in plasma membrane signal transduction, such as heterotrimeric G proteins, are also associated with membranes of different intracellular organelles.

Animals↗