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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 343 records · Page 19Linked to original sources
PubMed1986

Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system.

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.

Adenylyl Cyclase Inhibitors↗
PubMed1986

Mammalian alpha 1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit.

alpha 1-Adrenergic receptors from a cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl) pentanoyl]-1-piperazinyl]-quinazoline), an alpha 1 receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent Mr = 80,000 that co-migrates with the peptide labeled by the specific alpha 1-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[125I]iodophenyl)pentanoyl] -1-piperazinyl] quinazoline. The specific activity (approximately 13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of Mr = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate alpha 1-adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure alpha 1-adrenergic receptor and the pure human platelet alpha 2-adrenergic receptor (Regan, J.W., Nakata, H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology.

Animals↗
PubMed1986

Photoaffinity labeling of human platelet and rabbit kidney alpha 2-adrenoceptors with [3H]SKF 102229.

A newly developed alpha 2-adrenergic photoaffinity ligand, 3-methyl-6-chloro-9-azido-1H-2,3,4,5-tetrahydro-3-benzazepine (SKF 102229), has been radiolabeled with tritium to a specific activity of approximately 80 Ci/mmol. Using membranes prepared from human platelets and from rabbit kidney, alpha 2-adrenoceptors have been covalently labeled following photolysis in the presence of [3H]SKF 102229. As determined by SDS-PAGE, the apparent molecular weight of alpha 2-adrenoceptors from both of these tissues was 64,000. The yield of covalent insertion of [3H]SKF 102229 into the alpha 2-adrenoceptor was very good. Thus, following photolysis up to 90% of the alpha 2-adrenoceptors could be irreversibly labeled with [3H]SKF 102229.

Affinity Labels↗
PubMed1986

A novel catecholamine-activated adenosine cyclic 3',5'-phosphate independent pathway for beta-adrenergic receptor phosphorylation in wild-type and mutant S49 lymphoma cells: mechanism of homologous desensitization of adenylate cyclase.

Virtually all known biological actions stimulated by beta-adrenergic and other adenylate cyclase coupled receptors are mediated by cAMP-dependent protein kinase. Nonetheless, "homologous" or beta-adrenergic agonist-specific desensitization does not require cAMP. Since beta-adrenergic receptor phosphorylation may be involved in desensitization, we studied agonist-promoted receptor phosphorylation during homologous desensitization in wild-type S49 lymphoma cells (WT) and two mutants defective in the cAMP-dependent pathway of beta-agonist-stimulated protein phosphorylation (cyc- cannot generate cAMP in response to beta-adrenergic agonists; kin- lacks cAMP-dependent kinase). All three cell types demonstrate rapid, beta-adrenergic agonist-promoted, stoichiometric phosphorylation of the receptor which is clearly not cAMP mediated. The amino acid residue phosphorylated is solely serine. These data demonstrate, for the first time, that catecholamines can promote phosphorylation of a cellular protein (the beta-adrenergic receptor) via a cAMP-independent pathway. Moreover, the ability of cells with mutations in the adenylate cyclase-cAMP-dependent protein kinase pathway to both homologously desensitize and phosphorylate the beta-adrenergic receptors provides very strong support for the notion that receptor phosphorylation may indeed be central to the molecular mechanism of desensitization.

Adenosine Triphosphate↗
PubMed1986

Purification and characterization of the human platelet alpha 2-adrenergic receptor.

Human platelet alpha 2-adrenergic receptors have been purified approximately 80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall yield starting from the membranes is approximately 2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of Mr 64,000. The specific binding activity of the alpha 2-adrenergic receptor after four chromatographic steps is 14.5 nmol/mg protein. This value is consistent with the expected theoretical specific activity (15.6 nmol/mg) for a protein with a molecular mass of 64,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. The purified protein can be covalently labeled with the alkylating alpha-adrenergic ligand, [3H]phenoxybenzamine. This labeling is specific, and it shows that the Mr 64,000 protein contains the ligand binding site of the alpha 2-adrenergic receptor. In addition, the competitive binding of ligands to the purified receptor protein shows the proper alpha 2-adrenergic specificity. The alpha 2-adrenergic receptor contains an essential sulfhydryl residue. Thus, exposure of the purified receptor to the sulfhydryl-specific reagent, phenylmercuric chloride, resulted in an 80% loss of binding activity. This loss of binding activity was prevented when exposure to phenylmercuric chloride was done in the presence of alpha 2-adrenergic ligands, and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified alpha 2-adrenergic receptors was obtained with Staphylococcus aureus V-8 protease, alpha-chymotrypsin, and papain. In a comparison with purified beta 2-adrenergic receptors, no common partial proteolytic products were found.

Blood Platelets↗
PubMed1986

Functional reconstitution of the alpha 2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles.

We describe the successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles. The receptor is the alpha 2-adrenergic receptor (alpha 2AR) which has been partially purified (approximately 500-5000-fold) from human platelet membranes. The nucleotide-binding regulatory proteins include purified preparations of human erythrocyte Ni and Ns, bovine retinal transducin and the recently discovered bovine brain No. Addition of the physiologic ligand, epinephrine, to vesicles containing the alpha 2AR and Ni results in stimulation of the GTPase activity in Ni. This stimulation of GTPase activity by epinephrine is prevented in the presence of the alpha-adrenergic antagonist, phentolamine, which indicates that a functional reconstitution of the alpha 2AR and Ni has been established. The maximum turnover number for the alpha 2AR-mediated epinephrine-stimulated GTPase activity in Ni is similar to the maximal turnover numbers obtained for the beta-adrenergic receptor-mediated isoproterenol-stimulated GTPase activity in Ns and the rhodopsin-mediated light-stimulated GTPase activity in transducin (0.5-1.5 mol of Pi released per min per mol of nucleotide regulatory protein). Functional similarities between the alpha 2AR and rhodopsin are observed in their interactions with the various nucleotide-binding regulatory proteins. Thus, both of these receptor proteins are capable of promoting the maximal activation of Ni and No while being much less effective in promoting the activation of Ns. However, there are differences between the alpha 2AR and rhodopsin in their interactions with transducin. Specifically, while rhodopsin will maximally activate transducin, the alpha 2AR is much less effective in promoting this activation (i.e. approximately 20% as effective as rhodopsin). Overall, these results suggest the following specificities of interaction: for rhodopsin, transducin approximately equal to Ni approximately equal to No much greater than Ns; while for alpha 2AR, Ni approximately equal to No greater than transducin greater than or equal to Ns.

Adenylyl Cyclases↗
PubMed1986

Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes.

Incubation of intact frog erythrocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested beta-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the Vmax of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity (KD) or the binding capacity (Bmax) for the beta-adrenergic antagonist [125I]cyanopindolol. In addition, agonist/[125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E1-, forskolin-, NaF-, and MnCl2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly (t1/2 approximately equal to 5 min) and with an EC50 of about 10(-7) to 10(-6) M. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4 beta-phorbol 12,13-dibutyrate, 4 beta-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate, and 4-O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.

Adenylyl Cyclases↗
PubMed1986

Regulation of adrenergic receptor function by phosphorylation.

Mounting evidence suggests that the physiological function of the various subtypes of adrenergic receptors is controlled by phosphorylation/dephosphorylation reactions. It seems intuitively unlikely that this phenomenon will be limited simply to the adrenergic receptors, since these receptors share transmembrane signaling pathways with a host of other plasma membrane receptors. Different types of kinases appear to be involved. On the one hand, phosphorylation reactions may operate in a classical feedback regulatory sense. Thus, the cAMP-dependent protein kinase, once activated by a beta-agonist, can feedback-regulate the function of the receptors by phosphorylating and desensitizing them. Similarly, protein kinase C appears to be able to feedback-regulate the function of alpha 1-adrenergic receptors by phosphorylation. There may also be "cross talk" between the systems. Thus, protein kinase C, when stimulated by phorbols, is able to phosphorylate and desensitize the beta-adrenergic receptors. Moreover, very recently we have found that the cAMP-dependent protein kinase can phosphorylate the alpha 1-adrenergic receptors in vitro. These are examples of one transmembrane signaling system regulating the function of another. Perhaps most interestingly, it appears that there may be a previously unappreciated class of receptor kinases in the cytosol of cells. The first of these, which we have recently found and named beta-ARK, serves to phosphorylate only the agonist-occupied form of the beta-adrenergic receptor. As noted, it is somewhat analogous to the rhodopsin kinase. Such highly specific receptor kinases, which can phosphorylate only the agonist-occupied form of a receptor, represent a potentially elegant mechanism for controlling the function of receptors in a fashion which is linked to their physiological stimulation. How widespread such kinases are, and the actual roles which they play in regulating receptor function, remain to be determined. Finally, it should be stressed that although this review has focused on the regulatory role of receptor phosphorylation, it is by no means our intent to suggest that receptors are the only locus for physiological control of sensitivity to hormone and drug reaction. There is already evidence that guanine nucleotide regulatory proteins can be regulated, and it seems likely that each of the components of the system, including the adenylate cyclase, are likely to be involved in various forms of complex regulation. To date, however, the receptors represent that component of the system whose regulation we understand in the greatest detail.

Animals↗
PubMed1986

Ciba-Geigy award for outstanding research. Regulation of adrenergic receptor function by phosphorylation.

The various subtypes of adrenergic receptors represent distinct structural entities which are coupled in different ways to two major transmembrane signalling systems, the adenylate cyclase and phosphatidyl-inositol pathways. Recent evidence suggests that the functional linkage of both beta and alpha 1-adrenergic receptors to their respective effector systems is regulated by covalent modification of the receptors by phosphorylation-dephosphorylation reactions. Receptor phosphorylation appears to lead to desensitization of the biological response to receptor stimulation. Several kinases including protein kinase A, protein kinase C and a cAMP independent kinase appear to participate in these reactions.

Adenylate Kinase↗
PubMed1986

Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors.

beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta-AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist-promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors.

Alprostadil↗
PubMed1986

Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution.

Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.

Adenylyl Cyclases↗
PubMed1986

Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor.

Agonist-promoted desensitization of adenylate cyclase is intimately associated with phosphorylation of the beta-adrenergic receptor in mammalian, avian, and amphibian cells. However, the nature of the protein kinase(s) involved in receptor phosphorylation remains largely unknown. We report here the identification and partial purification of a protein kinase capable of phosphorylating the agonist-occupied form of the purified beta-adrenergic receptor. The enzyme is prepared from a supernatant fraction from high-speed centrifugation of lysed kin- cells, a mutant of S49 lymphoma cells that lacks a functional cAMP-dependent protein kinase. The beta-agonist isoproterenol induces a 5- to 10-fold increase in receptor phosphorylation by this kinase, which is blocked by the antagonist alprenolol. Fractionation of the kin- supernatant on molecular-sieve HPLC and DEAE-Sephacel results in a 50- to 100-fold purified beta-adrenergic receptor kinase preparation that is largely devoid of other protein kinase activities. The kinase activity is insensitive to cAMP, cGMP, cAMP-dependent kinase inhibitor, Ca2+-calmodulin, Ca2+-phospholipid, and phorbol esters and does not phosphorylate general kinase substrates such as casein and histones. Phosphate appears to be incorporated solely into serine residues. The existence of this novel cAMP-independent kinase, which preferentially phosphorylates the agonist-occupied form of the beta-adrenergic receptor, suggests a mechanism that may explain the homologous or agonist-specific form of adenylate cyclase desensitization. It also suggests a general mechanism for regulation of receptor function in which only the agonist-occupied or "active" form of the receptor is a substrate for enzymes inducing covalent modification.

Adrenergic beta-Agonists↗
PubMed1986

Identification of the subunit structure of rat pineal adrenergic receptors by photoaffinity labeling.

The adrenergic receptors of rat pineal gland were investigated using radiolabeled ligand binding and photoaffinity labeling techniques. 125I-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone (125I-HEAT) and 125I-cyanopindolol (125I-CYP) labeled specific sites on rat pineal gland membranes with equilibrium dissociation constants (KD) of 48 (+/- 5) pM and 30 (+/- 5) pM, respectively. Binding site maxima were 481 (+/- 63) and 1,020 (+/- 85) fmol/mg protein. The sites labeled by 125I-HEAT had the pharmacological characteristics of alpha 1-adrenergic receptors. 125I-CYP-labeled beta-adrenergic receptors were characterized as a homogeneous population of beta 1-adrenergic receptors. The alpha 1- and beta 1-adrenergic receptors were covalently labeled with the specific photoaffinity probes 4-amino-6,7-dimethoxy-2-(4-[5-(4-azido-3-[125I]iodophenyl) pentanoyl]-1-piperazinyl) quinazoline (125I-APDQ) and 125I-p-azidobenzylcarazolol (125I-pABC). 125I-APDQ labeled an alpha 1-adrenergic receptor peptide of Mr = 74,000 (+/- 4,000), which was similar to peptides labeled in rat cerebral cortex, liver, and spleen. 125I-pABC labeled a single beta 1-adrenergic receptor peptide with a Mr = 42,000 (+/- 1,500), which differed from the 60-65,000 peptide commonly seen in mammalian tissues. Possible reasons for these differences are discussed.

Affinity Labels↗
PubMed1985

Phorbol ester effects on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells.

Tumor promoting phorbol esters stimulate Ca++ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for 125I-HEAT binding to alpha 1-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate alpha 1-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating alpha 1-adrenergic receptor functional properties.

Animals↗
PubMed1985

Arylazide photoaffinity probe for alpha 2-adrenoceptors.

An arylazide photoaffinity probe for alpha 2-adrenoceptors has been developed and characterized. The compound, 3-methyl-6-chloro-9-azido-1H-2,3,4,5-tetrahydro-3-benzazepine (SKF 102229), had a Ki for the human platelet alpha 2-adrenoceptor of approximately 40 nM. Upon exposure to ultraviolet light, SKF 102229 irreversibly blocked the binding of [3H]yohimbine to both membrane bound and solubilized, partially purified, receptors. The extent of alpha 2-adrenoceptor blockade was dependent upon both the length of exposure to ultraviolet light and the concentration of SKF 102229. Typically, a 60% decrease in alpha 2-adrenoceptor number is obtained following 8 min of photolysis in the presence of 100 nM SKF 102229. The pharmacologic characteristics of the irreversible blockade produced by SKF 102229 were those of an alpha 2-adrenoceptor. Thus, photodependent, irreversible blockade of alpha 2-adrenoceptors by SKF 102229 was prevented by the concomitant presence of phentolamine or p-aminoclonidine but not by prazosin. Given its specificity and efficient blockade of the ligand binding site, SKF 102229 should prove useful for studies of the structure and function of alpha 2-adrenoceptors.

Absorption↗
PubMed1985

Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems.

The adenylate cyclase coupled inhibitory nucleotide regulatory protein (Ni) and the bovine retinal nucleotide regulatory protein transducin (T) appear to share some common functional properties since their GTPase activity is stimulated to similar extents by the retinal photoreceptor rhodopsin. In the present work, we sought to assess whether these functional similarities might extend to their interaction with adenylate cyclase. This necessitated the development of reconstitution systems in which guanine nucleotide regulatory protein mediated inhibition of adenylate cyclase activity could be demonstrated and characterized in a lipid milieu. In the absence of the pure human erythrocyte stimulatory nucleotide regulatory protein (Ns), the insertion into phospholipid vesicles of either pure Ni from human erythrocytes or pure bovine T with the resolved catalytic moiety of bovine caudate adenylate cyclase (C) does not establish GppNHp inhibition of either Mg2+- or forskolin-stimulated adenylate cyclase. However, the coinsertion into lipid vesicles of either Ni or T with Ns and resolved C results in an inhibition of Ns(GppNHp) stimulatable C activity. As is the case in intact membranes, the reconstituted inhibition of the Ns-stimulated C activity extends into the steady-state phase of time courses of activity. This inhibition is highly sensitive to the MgCl2 concentration. At 2 mM MgCl2, the inhibition is greater than 80% while at 50 mM MgCl2 it is only approximately 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1985

Phosphorylation of the mammalian beta-adrenergic receptor by cyclic AMP-dependent protein kinase. Regulation of the rate of receptor phosphorylation and dephosphorylation by agonist occupancy and effects on coupling of the receptor to the stimulatory guanine nucleotide regulatory protein.

In some systems, such as the turkey erythrocyte, agonist-promoted phosphorylation of the beta-adrenergic receptor appears to be associated with desensitization of the adenylate cyclase system. This process can be partially mimicked by cyclic AMP analogs. Accordingly, we have investigated the phosphorylation of the pure mammalian beta-adrenergic receptor by the pure catalytic subunit of the cyclic AMP-dependent protein kinase. The beta-adrenergic receptor, purified from hamster lung to apparent homogeneity, contains a single polypeptide of Mr approximately 64,000. The receptor can be phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase (approximately 2 mol of phosphate (on serine residues) per mol). Isoproterenol, a beta-agonist, promoted a 2-3-fold increase in the rate of receptor phosphorylation which was blocked by the beta-antagonists propranolol and alprenolol. High performance liquid chromatographic tryptic peptide mapping reveals two major phosphorylation sites. Phosphorylated receptor can be completely dephosphorylated by a high molecular weight phosphoprotein phosphatase. The rate of receptor dephosphorylation is enhanced 2-3-fold by isoproterenol and this effect is blocked by alprenolol. The functional significance of receptor phosphorylation was examined using ligand binding and reconstitution techniques. While the binding of isoproterenol and alprenolol to the receptor was unaffected by phosphorylation, the ability of the receptor to interact with the stimulatory guanine nucleotide regulatory protein, as assessed by isoproterenol-promoted GTPase activity, was decreased 24 +/- 1% (mean +/- S.E., p less than 0.001, n = 17). The quantitative extent of receptor phosphorylation and functional impairment are virtually identical to those previously observed when intact turkey erythrocytes were incubated with cyclic AMP. These data provide a direct demonstration of regulation of the function of the isolated beta-adrenergic receptor by cyclic AMP-dependent protein kinase.

Animals↗