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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 235 records · Page 13Linked to original sources
PubMed1991

The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase.

Light-dependent deactivation of rhodopsin as well as homologous desensitization of beta-adrenergic receptors involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase (RK) and beta-adrenergic receptor kinase (beta ARK), respectively. We report here the cloning of a complementary DNA for RK. The deduced amino acid sequence shows a high degree of homology to beta ARK. In a phylogenetic tree constructed by comparing the catalytic domains of several protein kinases, RK and beta ARK are located on a branch close to, but separate from the cyclic nucleotide-dependent protein kinase and protein kinase C subfamilies. From the common structural features we conclude that both RK and beta ARK are members of a newly delineated gene family of guanine nucleotide-binding protein (G protein)-coupled receptor kinases that may function in diverse pathways to regulate the function of such receptors.

Amino Acid Sequence↗
PubMed1991

Cyclic phosphorylation-dephosphorylation of rhodopsin in retina by protein kinase FA (the activator of ATP.Mg-dependent protein phosphatase).

The ATP.Mg-dependent protein phosphatase activating factor (protein kinase FA) was identified to exist in bovine retina. Furthermore, rhodopsin, the visual light pigment associated with rod outer segments in retina, could be well phosphorylated by kinase FA to about 0.9 mol of phosphates per mol of protein. Moreover, more than 90% of the phosphates in [32P]-rhodopsin could be completely removed by ATP.Mg-dependent protein phosphatase and the rhodopsin phosphatase activity was strictly kinase FA-dependent. Taken together, the results provide initial evidence that a cyclic phosphorylation-dephosphorylation of rhodopsin can be controlled by the retina-associated protein kinase FA, representing an efficient cyclic cascade mechanism possibly involved in the rapid regulation of rhodopsin function in retina.

Adenosine Triphosphate↗
PubMed1991

Cloning, expression, and chromosomal localization of beta-adrenergic receptor kinase 2. A new member of the receptor kinase family.

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied form of the beta-adrenergic and related G protein-coupled receptors. Structural features of this enzyme have been elucidated recently by the isolation of a cDNA that encodes bovine beta ARK. Utilizing a catalytic domain fragment of the beta ARK cDNA to screen a bovine brain cDNA library we have isolated a clone encoding a beta ARK-related enzyme which we have termed beta ARK2. Overall, this enzyme has 85% amino acid identity with beta ARK, with the protein kinase catalytic domain having 95% identity. The ability of beta ARK2 to phosphorylate various substrates was studied after expression in COS 7 cells. Although beta ARK2 is essentially equiactive with beta ARK in phosphorylating an acid-rich synthetic model peptide it was only approximately 50% as active when the substrate was the agonist-occupied beta 2-adrenergic receptor and only approximately 20% as active toward light-bleached rhodopsin. As with beta ARK, phosphorylation of the receptor substrates by beta ARK2 was completely stimulus dependent. RNA blot analysis with selected bovine tissues reveals an mRNA of 8 kilobases with a distribution similar to that of beta ARK. More detailed RNA analysis using a ribonuclease protection assay in various rat tissues suggests that the beta ARK2 message is present at much lower levels (typically 10-20%) than the beta ARK message. In the rat the beta ARK2 mRNA is localized predominantly in neuronal tissues although low levels are also observed in various peripheral tissues. The beta ARK2 gene has been localized to a region of mouse chromosome 5 whereas the beta ARK gene is localized on mouse chromosome 19. These data suggest the existence of a "family" of receptor kinases which may serve broadly to regulate receptor function.

Amino Acid Sequence↗
PubMed1991

Comparative rates of desensitization of beta-adrenergic receptors by the beta-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase.

Three separate processes may contribute to rapid beta-adrenergic receptor desensitization: functional uncoupling from the stimulatory guanine nucleotide-binding protein Gs, mediated by phosphorylation of the receptors by two distinct kinases, the specific beta-adrenergic receptor kinase (beta ARK) and the cyclic AMP-dependent protein kinase A (PKA), as well as a spatial uncoupling via sequestration of the receptors away from the cell surface. To evaluate the relative importance and potential role of the various processes in different physiological situations, a kinetic analysis of these three mechanisms was performed in permeabilized A431 epidermoid carcinoma cells. To allow a separate analysis of each mechanism, inhibitors of the various desensitization mechanisms were used: heparin to inhibit beta ARK, the PKA inhibitor peptide PKI to inhibit PKA, and concanavalin A treatment to prevent sequestration. Isoproterenol-induced phosphorylation of beta 2 receptors in these cells by beta ARK occurred with a t1/2 of less than 20 sec, whereas phosphorylation by PKA had a t1/2 of about 2 min. Similarly, beta ARK-mediated desensitization of the receptors proceeded with a t1/2 of less than 15 sec, and PKA-mediated desensitization with a t1/2 of about 3.5 min. Maximal desensitization mediated by the two kinases corresponded to a reduction of the signal-transduction capacity of the receptor/adenylyl cyclase system by about 60% in the case of beta ARK and by about 40% in the case of PKA. Receptor sequestration was much slower (t1/2 of about 10 min) and involved no more than 30% of the cell surface receptors. It is concluded that beta ARK-mediated phosphorylation is the most rapid and quantitatively most important factor contributing to the rapid desensitization. This rapidity of the beta ARK-mediated mechanism makes it particularly well suited to regulate beta-adrenergic receptor function in rapidly changing environments such as the synaptic cleft.

Carcinoma, Squamous Cell↗
PubMed1991

Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase.

The beta-adrenergic receptor kinase (beta-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by beta-ARK are poorly defined, the identification of substrate amino acids that are critical to phosphorylation by the kinase are also unknown. In this study, a peptide whose sequence is present in a portion of the third intracellular loop region of the human platelet alpha 2-adrenergic receptor is shown to serve as a substrate for beta-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this alpha 2-peptide resulted in a complete loss of phosphorylation by the kinase. A family of peptides was synthesized to further study the role of acidic amino acids in peptide substrates of beta-ARK. By kinetic analyses of the phosphorylation reactions, beta-ARK exhibited a marked preference for negatively charged amino acids localized to the NH2-terminal side of a serine or threonine residue. While there were no significant differences between glutamic and aspartic acid residues, serine-containing peptides were 4-fold better substrates than threonine. Comparing a variety of kinases, only rhodopsin kinase and casein kinase II exhibited significant phosphorylation of the acidic peptides. Unlike beta-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to beta-ARK and RK was a much greater Km for peptide substrates as compared to that for intact receptor substrates.

Amino Acid Sequence↗
PubMed1991

cDNA cloning and chromosomal localization of the human beta-adrenergic receptor kinase.

The beta-adrenergic receptor kinase (beta ARK) mediates agonist-dependent phosphorylation of the beta 2-adrenergic and related G protein-coupled receptors. A cDNA encoding bovine beta ARK has recently been isolated. In this work we have isolated a cDNA encoding human beta ARK from a retinal cDNA library. The cDNA encodes a protein of 689 amino acids with an overall 98.0% amino acid and 92.5% nucleotide identity with bovine beta ARK. Chromosomal location of the human beta ARK gene was determined by correlating the presence of the beta ARK locus with retention of a specific human chromosome in a rodent-human hybrid panel. This analysis revealed that the human beta ARK locus segregated with the long arm of chromosome 11, centromeric to 11q13.

Amino Acid Sequence↗
PubMed1991

A small region of the beta-adrenergic receptor is selectively involved in its rapid regulation.

Plasma membrane receptors that couple to guanine nucleotide-binding regulatory proteins (G proteins) undergo a variety of rapid (minutes) and longer term (hours) regulatory processes induced by ligands. For the beta 2-adrenergic receptor (beta 2AR), the rapid processes include functional desensitization, mediated by phosphorylation of the receptor by the cAMP-dependent protein kinase and the beta-adrenergic receptor kinase, as well as a loss of hydrophilic ligand binding proposed to represent sequestration of receptors into a cellular compartment distinct from the plasma membrane. The slower processes include beta 2AR down-regulation (i.e., a decrease in the total cellular complement of receptors). It is not yet known whether beta 2AR phosphorylation and/or sequestration are prerequisites for down-regulation of the receptor. Like other G protein-coupled receptors, the beta 2AR molecule spans the plasma membrane seven times, and the cytoplasmic carboxyl-terminal domain has been proposed to contain molecular determinants for each of these regulatory processes. We replaced four serine and threonine residues located within a 10-amino acid segment of this domain of beta 2AR and thereby prevented agonist-promoted phosphorylation, sequestration, and rapid desensitization of the adenylyl cyclase response. In contrast, these mutations did not affect functional coupling to the stimulatory G protein Gs or long-term down-regulation. These findings thus define a small, hitherto unappreciated region of the receptor molecule that may selectively subserve its rapid regulation. In addition, with the demonstration that beta 2AR does not have to be phosphorylated or sequestered in order to enter the down-regulation pathway, the results suggest that the classical receptor endocytosis model may not be appropriate for beta 2AR regulation.

Adenylyl Cyclases↗
PubMed1991

Molecular cloning and expression of the cDNA for the alpha 1A-adrenergic receptor. The gene for which is located on human chromosome 5.

Pharmacological and molecular cloning studies have demonstrated heterogeneity of alpha 1-adrenergic receptors. We have now cloned two alpha 1-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster alpha 1B-adrenergic receptor as a probe. The deduced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an alpha 1-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster alpha 1B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 1-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenylephrine and methoxamine suggests that cDNA RA42 encodes the alpha 1A receptor subtype. Northern blot analysis of various rat tissues also shows the distribution expected of the alpha 1A receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second alpha 1-adrenergic receptor cloned represents the rat homolog of the hamster alpha 1B subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evidence for the existence of three alpha 1-adrenergic receptor subtypes.

Amino Acid Sequence↗
PubMed1991

Functional interactions of recombinant alpha 2 adrenergic receptor subtypes and G proteins in reconstituted phospholipid vesicles.

The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.

Animals↗
PubMed1991

Coupling of a mutated form of the human beta 2-adrenergic receptor to Gi and Gs. Requirement for multiple cytoplasmic domains in the coupling process.

We constructed five genes encoding mutant human beta 2-adrenergic receptor sequence (beta 2AR) which contained 12-22 amino acid substitutions with corresponding sequence from the human alpha 2AAR in order to assess the receptor domains involved in Gs versus Gi recognition and coupling. Mutant beta 2AR with substitutions in the N (S1)- and C-terminal (S2) portions of the third intracellular loop, the proximal cytoplasmic tail (S3), and two combinations thereof (S2,3 and S1,2,3), were stably expressed in Chinese hamster fibrobasts (CHW-1102), as were the human beta 2AR and alpha 2AAR at comparable receptor levels. All mutant receptors with S2 substitutions (i.e. S2, S2,3, S1,2,3) were significantly (approximately 85%) uncoupled from Gs. Upon exposure to pertussis toxin, which uncouples receptors from Gi, S1,2,3 exhibited a 526 +/- 99% increase in agonist-stimulated adenylylcyclase activity compared with a 59 +/- 13% increase with the wild type receptor. This enhanced ability of S1,2,3 to interact with Gs following pertussis toxin treatment indicates that, in the absence of toxin exposure, substantial coupling occurs between the mutant receptor and Gi. Mutant beta 2AR bearing only one or two alpha 2AAR-substituted sequences showed no such enhancement. Forskolin-stimulated enzyme activities were increased by pertussis toxin treatment to similar degrees in all clones examined, indicating that the observed effects are confined to the receptor-mediated pathway. In the absence of GTP, competition binding experiments with S1,2,3, beta 2AR and alpha 2AAR revealed that approximately 40-50% of the receptors formed a high affinity binding state for agonist. Pertussis toxin treatment markedly reduced this to approximately 19% with S1,2,3, while having no effect on beta 2AR and completely eliminating high affinity agonist binding to alpha 2AAR. These results suggest that S1,2,3 interacts with Gi as well as Gs, and that receptor:G protein coupling requires the concerted participation of multiple cytoplasmic receptor domains.

Adenosine Diphosphate Ribose↗
PubMed1991

5-HT1A and histamine H1 receptors in HeLa cells stimulate phosphoinositide hydrolysis and phosphate uptake via distinct G protein pools.

Regulation of phosphate uptake was studied in a HeLa cell line after transfection with DNA encoding the human 5-HT1A receptor. In these cells, 5-HT stimulates sodium-dependent phosphate uptake via protein kinase C activation. Endogenous histamine H1 receptors (739 +/- 20 fmol/mg protein) were identified with [3H]pyrilamine. Histamine (i) stimulated phosphoinositide hydrolysis (EC50 = 8.6 +/- 4.1 microM), (ii) activated protein kinase C (2.4-fold increase in activity), and (iii) increased phosphate uptake (EC50 = 3.2 +/- 1.8 microM) by increasing maximal transport (Vmax(basal) = 6.2 +/- 0.3 versus Vmax(histamine) = 9.1 +/- 0.4) without changing the affinity of the transport process for phosphate. Prolonged treatment with 16 microM phorbol 12-myristate 13-acetate completely blocked protein kinase C activation and markedly attenuated the stimulation of phosphate uptake induced by histamine, establishing that 5-HT and histamine stimulate phosphate uptake through the common pathway of protein kinase C activation. The linkages of the histamine H1 and 5-HT1A receptors to G protein pools were assessed in two ways. (i) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake associated with histamine were insensitive to pertussis toxin, whereas those associated with 5-HT were very sensitive to pertussis toxin. (ii) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake induced by histamine and 5-HT were additive. These findings suggest that distinct receptor types can stimulate phosphoinositide hydrolysis, protein kinase C, and phosphate uptake in an additive fashion through distinct pools of G proteins in a single cell type.

Biological Transport↗
PubMed1991

Genomic organization of adrenergic and serotonin receptors in the mouse: linkage mapping of sequence-related genes provides a method for examining mammalian chromosome evolution.

Five sequence-related genes encoding four adrenergic receptors and a serotonin receptor were localized to specific regions of four mouse chromosomes with respect to 11 other genetic markers. Linkage was established by the analysis of the haplotypes of 114 interspecific backcross mice. Adra2r (alpha 2-C10) and Adrb1r (beta 1) receptors mapped to the distal region of mouse chromosome 19. These genes were separated by 2.6 +/- 1.5 cM in a segment of mouse chromosome 19 that has a similar organization of these genes on the long arm of human chromosome 10. The Adra1r (alpha 1B), Adrb2r (beta 2), and Htra1 (5HT1A) genes mapped to proximal mouse chromosome 11, proximal mouse chromosome 18, and distal mouse chromosome 13, respectively. The organization of genes linked to these loci on regions of the three mouse chromosomes is consistent with the organization of homologous human genes on human chromosome 5. These findings further define the relationship of linkage groups conserved during the evolution of the mouse and human genomes. We have identified a region that may have been translocated during evolution and suggest that the human genomic organization of adrenergic receptors more closely resembles that of a putative primordial ancestor.

Animals↗
PubMed1991

Dual coupling of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same Gi protein.

The cloned 5-HT1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects of 5-hydroxytryptamine (5-HT) to inhibit cAMP formation and to stimulate the hydrolysis of phosphatidylinositol. Both responses were found to be pertussis toxin sensitive. We have examined these two responses in membranes derived from these cells and show that the 5-HT1A receptor can directly regulate the activity of adenylyl cyclase and phospholipase C in response to agonist. In order to examine whether the same or distinct guanine nucleotide-binding regulatory protein(s) (G protein) are involved in these two signal transduction pathways, we used anti-peptide antibodies recognizing the alpha-subunits of Gi1, Gi2, Gi3 as specific tools, since these pertussis toxin substrates are expressed in HeLa cells. These antibodies have previously been shown to prevent receptor-G protein coupling by binding to the regions of G proteins which are putatively involved in interaction with receptors. Our results indicate that the Gi proteins, but preferentially Gi3, mediate the effects of 5-HT both to inhibit adenylyl cyclase and to stimulate phospholipase C. These findings demonstrate that the same receptor interacting with the same G protein can regulate several distinct effector molecules.

Adenylate Cyclase Toxin↗