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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 289 records · Page 16Linked to original sources
PubMed1989

Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet alpha 2-adrenergic receptor.

The human platelet alpha 2-adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known [Kobilka et al. (1987) Science 238, 650-656]. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, we have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [3H]SKF 102229 (an antagonist) or p-azido[3H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [3H]SKF 102229 labeled receptor yielded one peptide of Mr 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of Mr 4000, which was further digested to the Mr 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[3H]clonidine-labeled receptor, a similar Mr 2400 peptide was obtained by lysylendopeptidase cleavage. This Mr 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet alpha 2-adrenergic receptor.

Affinity Labels↗
PubMed1989

Inhibition of the beta-adrenergic receptor kinase by polyanions.

The beta-adrenergic receptor kinase, which specifically phosphorylates the agonist-occupied beta-adrenergic receptor, is strongly inhibited by polyanions. Heparin and dextran sulfate inhibit the enzyme with an IC50 of approximately 0.15 microM. De-N-sulfated heparin is approximately 8-fold less potent. Other acid mucopolysaccharides such as heparan sulfate and chondroitin sulfates B and C are also less effective. Polyaspartic and polyglutamic acid also inhibit with IC50 values of 1.3-2 microM. Inositol hexasulfate, with an IC50 of 13 microM is approximately 270-fold more potent than inositol hexaphosphate implicating the sulfate group as a major determinant of the inhibition. The inhibition by heparin is competitive with substrate and of mixed type with respect to ATP. Polycations also inhibit receptor phosphorylation by beta-adrenergic receptor kinase. Polylysine is more effective with an IC50 of 69 microM, while spermine (990 microM) and spermidine (2570 microM) are less potent. Polylysine, spermine, and spermidine are also able to block effectively the inhibition by heparin. The identification of compounds which specifically inhibit beta-adrenergic receptor kinase should prove useful in further defining the biological role of this enzyme.

Animals↗
PubMed1989

Adrenergic receptor homologies in vertebrate and invertebrate species examined by DNA hybridization.

The deduced protein sequences of the mammalian adrenergic receptors (ARs) suggest that these proteins have evolved by several ancient gene duplication events. To investigate in what species these events may have occurred DNA fragments encoding the family of adrenergic receptors from human (beta 1AR and alpha 2AR) and hamster (beta 2AR and alpha 1AR) were used to detect homologous sequences in other vertebrates, invertebrates and unicellular organisms by Southern blot hybridization analysis. Sequences homologous to hamster beta 2AR were detected in lower vertebrates, invertebrates and Dictyostelium, but not in yeast or bacteria. Within vertebrates, sequences strongly homologous to human beta 1AR and human platelet alpha 2AR were confined to the higher vertebrates only. In the invertebrates, only Drosophila contained sequences homologous to hamster alpha 1AR. Our results suggest that non-mammalian species may contain receptors homologous to the mammalian adrenergic receptors and that the sequences homologous to human beta 2AR have been the most strongly conserved.

Animals↗
PubMed1989

cAMP stimulates transcription of the beta 2-adrenergic receptor gene in response to short-term agonist exposure.

In addition to conveying cellular responses to an effector molecule, receptors are often themselves regulated by their effectors. We have demonstrated that epinephrine modulates both the rate of transcription of the beta 2-adrenergic receptor (beta 2AR) gene and the steady-state level of beta 2AR mRNA in DDT1MF-2 cells. Short-term (30 min) exposure to epinephrine (100 nM) stimulates the rate of beta 2AR gene transcription, resulting in a 3- to 4-fold increase in steady-state beta 2AR mRNA levels. These effects are mimicked by 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) or foskolin but not by phorbol esters. The half-life of the beta 2AR mRNA after addition of actinomycin D (46.7 +/- 10.2 min; mean +/- SEM; n = 5) remained unchanged after 30 min of epinephrine treatment (46.8 +/- 10.6 min; mean +/- SEM; n = 4), indicating that a change in transcription rate is the predominant factor responsible for the increase of beta 2AR mRNA. Whereas brief exposure to epinephrine or Bt2cAMP does not significantly affect the total number of cellular beta 2ARs (assessed by ligand binding), continued exposure results in a gradual decline in beta 2AR number to approximately 20% (epinephrine) or approximately 45% (Bt2cAMP) of the levels in control cells by 24 hr. Similar decreases in agonist-stimulated adenylyl cyclase activity are observed. This loss of receptors with prolonged agonist exposure is accompanied by a 50% reduction in beta 2AR mRNA. Transfection of the beta 2AR promoter region cloned onto a reporter gene (bacterial chloramphenicol acetyltransferase) allowed demonstration of a 2- to 4-fold induction of transcription by agents that elevate cAMP levels, such as forskolin or phosphodiesterase inhibitors. These results establish the presence of elements within the proximal promoter region of the beta 2AR gene responsible for the transcriptional enhancing activity of cAMP and demonstrate that beta 2AR gene expression is regulated by a type of feedback mechanism involving the second messenger cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Inhibition of beta-adrenergic receptor kinase prevents rapid homologous desensitization of beta 2-adrenergic receptors.

Homologous (agonist-specific) desensitization of beta-adrenergic receptors (beta ARs) is accompanied by and appears to require phosphorylation of the receptors. We have recently described a novel protein kinase, beta AR kinase, which phosphorylates beta ARs in vitro in an agonist-dependent manner. This kinase is inhibited by two classes of compounds, polyanions and synthetic peptides derived from the beta 2-adrenergic receptor (beta 2AR). In this report we describe the effects of these inhibitors on the process of homologous desensitization induced by the beta-adrenergic agonist isoproterenol. Permeabilization of human epidermoid carcinoma A431 cells with digitonin was used to permit access of the charged inhibitors to the cytosol; this procedure did not interfere with the pattern of isoproterenol-induced homologous desensitization of beta 2AR-stimulated adenylyl cyclase. Inhibitors of beta AR kinase markedly inhibited homologous desensitization of beta 2ARs in the permeabilized cells. Inhibition of desensitization by heparin, the most potent of the polyanion inhibitors of beta AR kinase, occurred over the same concentration range (5-50 nM) as inhibition of purified beta AR kinase assessed in a reconstituted system. Inhibition of desensitization by heparin was accompanied by a marked reduction of receptor phosphorylation in the permeabilized cells. Whereas inhibitors of beta AR kinase inhibited homologous desensitization, inhibitors of protein kinase C and of cyclic-nucleotide-dependent protein kinases were ineffective. These data establish that phosphorylation of beta ARs by beta AR kinase is an essential step in homologous desensitization of the receptors. They further suggest a potential therapeutic value of inhibitors of beta AR kinase in inhibiting agonist-induced desensitization.

Animals↗
PubMed1989

Structure of the adrenergic and related receptors.

The isolation and sequencing of a number of G protein-coupled receptors has now provided extensive primary structure information for this family of homologous proteins. The diverse nature of these receptors suggests that the family of proteins may grow to include receptors for many neurotransmitters and perhaps many peptide hormones. The topography of these receptors, a single polypeptide with seven transmembrane segments, appears to have features well suited for the transmission of signals, via conformational changes, to the interior of the cell. Detailed site-directed mutagenesis studies are now underway in many laboratories to understand the significance of the topography and also the regions of homology evident in the structures of all of these receptors. Obvious features of interest are the precise residues involved in the coupling of the receptors to the G-proteins and the identification of the residues required for ligand binding in each of the receptors, as well as domains of these receptors involved in the regulation of receptor function. In addition, the availability of molecular probes for this family of proteins will permit the elucidation of mechanisms of regulation at the gene level.

Amino Acid Sequence↗
PubMed1989

Genetic regulation of beta-adrenergic receptors.

Several members of the family of receptors coupled to G proteins have been cloned in recent years. From the primary sequence information furnished by these clones, a characteristic seven-membrane spanning topography has emerged as the prototype for this class of receptors, in analogy with the opsin visual pigments. Cloned genes for the various receptors provide important tools for probing the regulation of their expression. For example, the underlying genetic basis for the expression of adrenergic receptor subtypes can now be explored. The physiological regulation of receptors by heterologous hormones, such as steroids and thyroid hormones, has long been suspected to involve changes in the expression of the relevant adrenergic receptor genes. At least for the glucocorticoids, beta 2AR expression is controlled at the level of transcription. The hormonal regulation of other adrenergic receptor subtypes is currently being explored.

Animals↗
PubMed1989

Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe.

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1989

Properties of the beta 1- and beta 2-adrenergic receptor subtypes revealed by molecular cloning.

The beta 1- and beta 2-adrenergic receptor subtypes are biochemically and functionally similar, because both receptors mediate the catecholamine-dependent activation of adenylate cyclase through the GTP-binding protein, Gs. Pharmacologically, the two receptors can be distinguished on the basis of their relative affinities for the agonists epinephrine and norepinephrine as well as their affinities for several selective antagonists. The primary structures of the human beta 1- and beta 2-adrenergic receptors have recently been deduced from the cloning of their genes and (or) cDNAs, revealing high sequence homology and a membrane topography of seven putative transmembrane regions similar to that of rhodopsin. Chimeric beta 1/beta 2-adrenergic receptor cDNAs have been constructed by site-directed mutagenesis and the chimeric RNA transcripts expressed in Xenopus laevis oocytes. The pharmacological properties of the expressed chimeric receptor proteins were assessed by radioligand binding utilizing subtype-selective agonists and antagonists. Apparently, several of the putative transmembrane regions contribute significantly to the determination of subtype selectivity, presumably by formation of a ligand-binding pocket, with determinants for agonist and antagonist binding being distinguishable.

Animals↗
PubMed1989

Altered patterns of agonist-stimulated cAMP accumulation in cells expressing mutant beta 2-adrenergic receptors lacking phosphorylation sites.

As with many other receptor-effector systems, the responsiveness of the beta-adrenergic receptor (beta AR)/adenylyl cyclase system undergoes desensitization upon agonist exposure. Phosphorylations of the receptor by the cAMP-dependent protein kinase (protein kinase A) and the beta AR kinase appear to play roles in such desensitization phenomena, but the functional significance of the receptor phosphorylation in intact cells has not been previously assessed. In this study, we constructed and expressed in a mammalian fibroblast line the normal (wild type) human beta 2 AR and mutant forms of the receptor that lack the putative phosphorylation sites for these two protein kinases. The two consensus sequences for phosphorylation by protein kinase A were altered by changing serines 261, 262 and 345, 346 to alanines. In another mutant, the 11 serines and threonines at the carboxy terminus of the protein that constitute the putative beta AR kinase phosphorylation sites were changed to alanines or glycines. The mutated receptors did not differ from the wild type in their affinities for agonists or antagonists or in their ability to mediate agonist stimulation of adenylyl cyclase. Moreover, their levels of expression in the cultured cells were the same. When stimulated with the beta AR agonist isoproterenol, cells bearing either the wild type or mutant receptors generated cAMP at essentially identical rates for the first 2 min. Cells bearing wild type receptors then showed a rapid desensitization characterized by a markedly diminished rate of cAMP production after the first few minutes of stimulation. However, cells bearing either of the mutated forms of the receptor showed much less desensitization and continued to generate cAMP at a rate 3-4 times greater than that observed in cells expressing the wild type receptor. In contrast, intact cell cAMP levels stimulated by prostaglandin E1 and forskolin were not different between cells bearing wild type or mutant beta AR. These results suggest an important physiological role for phosphorylation of the beta AR in regulating rapid agonist-induced desensitization in intact cells.

Amino Acid Sequence↗
PubMed1988

Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling.

Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human beta 2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human beta 2-adrenergic receptor. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.

Amino Acid Sequence↗
PubMed1988

The genomic clone G-21 which resembles a beta-adrenergic receptor sequence encodes the 5-HT1A receptor.

The recent cloning of the complementary DNAs and/or genes for several receptors linked to guanine nucleotide regulatory proteins including the adrenergic receptors (alpha 1, alpha 2A, alpha 2B, beta 1, beta 2), several subtypes of the muscarinic cholinergic receptors, and the visual 'receptor' rhodopsin has revealed considerable similarity in the primary structure of these proteins. In addition, all of these proteins contain seven putative transmembrane alpha-helices. We have previously described a genomic clone, G-21, isolated by cross-hybridization at reduced stringency with a full length beta 2-adrenergic receptor probe. This clone contains an intronless gene which, because of its striking sequence resemblance to the adrenergic receptors, is presumed to encode a G-protein-coupled receptor. Previous attempts to identify this putative receptor by expression studies have failed. We now report that the protein product of the genomic clone, G21, transiently expressed in monkey kidney cells has all the typical ligand-binding characteristics of the 5-hydroxytryptamine (5-HT1A) receptor.

Animals↗
PubMed1988

Beta-adrenergic receptors in hamster smooth muscle cells are transcriptionally regulated by glucocorticoids.

Steroid hormones modulate adrenergic receptor responsiveness and receptor number. To investigate the regulation of the beta 2-adrenergic receptor gene by glucocorticoids we examined the effects of the synthetic glucocorticoid agonist triamcinolone acetonide on the expression of beta 2-adrenergic receptors in DDT1MF-2 hamster smooth muscle cells. Glucocorticoid treatment (1 X 10(-7) M) produced a 2.2 +/- 0.4-fold (n = 8) increase in beta 2-adrenergic receptor number (maximum) between 6 and 12 h) as determined by radioligand binding and a similar increase in catecholamine-stimulated adenylate cyclase activity. Steady-state levels of beta 2-adrenergic receptor mRNA, analyzed by Northern blot hybridization, were increased 2.4 +/- 0.4-fold (n = 6) within 1 h, while actin mRNA levels were unchanged throughout the experiment. These steroid-induced increases in beta 2-adrenergic receptor mRNA returned to control levels by 24 h and were followed by a much slower decline in beta 2-adrenergic receptor in plasma membranes. The rate of beta 2-adrenergic receptor gene transcription, assessed by nuclear run-off transcription assays, increased 3.1 +/- 0.1-fold (n = 2) in cells treated for 30 min with 1 X 10(-7) M triamcinolone acetonide. These studies indicate that glucocorticoids regulate the beta 2-adrenergic receptor-adenylate cyclase system by controlling the rate of transcription of the beta 2-adrenergic receptor gene and hence the responsiveness of the enzyme to catecholamine stimulation.

Adenylyl Cyclases↗
PubMed1988

Dephosphorylation of the beta 2-adrenergic receptor and rhodopsin by latent phosphatase 2.

Recent evidence suggests that the function of receptors coupled to guanine nucleotide regulatory proteins may be controlled by highly specific protein kinases, e.g. rhodopsin kinase and the beta-adrenergic receptor kinase. In order to investigate the nature of the phosphatases which might be involved in controlling the state of receptor phosphorylation we studied the ability of four highly purified well characterized protein phosphatases to dephosphorylate preparations of rhodopsin or beta 2-adrenergic receptor which had been highly phosphorylated by beta-adrenergic receptor kinase. These included: type 1 phosphatase, calcineurin phosphatase, type 2A phosphatase, and the high molecular weight latent phosphatase 2. Under conditions in which all the phosphatases could dephosphorylate such common substrates as [32P]phosphorylase a and [32P]myelin basic protein at similar rates only the latent phosphatase 2 was active on the phosphorylated receptors. Moreover, a latent phosphatase activity was found predominantly in a sequestered membrane fraction of frog erythrocytes. This parallels the distribution of a beta-adrenergic receptor phosphatase activity recently described in these cells (Sibley, D. R., Strasser, R. H., Benovic, J. L., Daniel, K., and Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9408-9412). These data suggest a potential role for the latent phosphatase 2 as a specific receptor phosphatase.

Animals↗
PubMed1988

Regulation of the beta 2-adrenergic receptor and its mRNA in the rat ventral prostate by testosterone.

To investigate the regulation of beta 2AR expression in rat ventral prostate the effects of castration and testosterone replacement on the beta 2AR were studied by ligand binding and Northern blot analysis. Orchidectomy depressed beta 2AR number by 50% within 4 days and testosterone administration to 4-day castrates produced a rapid and complete recovery of beta 2AR number within 24 h. In contrast to receptor number, beta 2AR mRNA levels did not change relative to total RNA following castration. However, during the testosterone replacement period beta 2AR mRNA levels rose transiently, reaching a maximum (3.5-fold) between 8 and 12 h, and this increase in mRNA preceded the recovery in beta 2AR number in the membrane. Regulation of beta 2AR gene expression by testosterone in the ventral prostate is thus complex and probably involves both transcriptional and post-transcriptional components.

Animals↗
PubMed1988

Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity.

The alpha 2 and beta 2 adrenergic receptors, both of which are activated by epinephrine, but which can be differentiated by selective drugs, have opposite effects (inhibitory and stimulatory) on the adenylyl cyclase system. The two receptors are homologous with each other, rhodopsin, and other receptors coupled to guanine nucleotide regulatory proteins and they contain seven hydrophobic domains, which may represent transmembrane spanning segments. The function of specific structural domains of these receptors was determined after construction and expression of a series of chimeric alpha 2-,beta 2-adrenergic receptor genes. The specificity for coupling to the stimulatory guanine nucleotide regulatory protein lies within a region extending from the amino terminus of the fifth hydrophobic domain to the carboxyl terminus of the sixth. Major determinants of alpha 2- and beta 2-adrenergic receptor agonist and antagonist ligand binding specificity are contained within the seventh membrane spanning domain. Chimeric receptors should prove useful for elucidating the structural basis of receptor function.

Amino Acid Sequence↗
PubMed1988

Removal of phosphorylation sites from the beta 2-adrenergic receptor delays onset of agonist-promoted desensitization.

Eukaryotic cells have evolved a variety of mechanisms for dampening their responsiveness to hormonal stimulation in the face of sustained activation. The mechanisms for such processes, collectively referred to as desensitization, often involve alterations in the properties and number of cell-surface hormone receptors. It has been speculated that phosphorylation-dephosphorylation reactions, which are known to regulate the catalytic activities of enzymes, also regulate the function of receptors. Highly specific receptor kinases, such as rhodopsin kinase and beta-adrenergic receptor kinase, which show stimulus-dependent phosphorylation of receptors have been described. Direct evidence for a causal relationship between receptor phosphorylation and desensitization has been lacking however. Here we report that prevention of agonist-stimulated beta 2-adrenergic receptor (beta 2AR) phosphorylation by truncation of its serine and threonine-rich phosphate acceptor segment delays the onset of desensitization. We also show that selective replacement of these serine and threonine residues by alanine and glycine delays desensitization even further. These data provide the first direct evidence that one molecular mechanism of desensitization of G-protein-coupled receptors involves their agonist-induced phosphorylation.

Amino Acid Sequence↗