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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 361 records · Page 20Linked to original sources
PubMed1985

Homologous desensitization of beta-adrenergic receptor coupled adenylate cyclase. Resensitization by polyethylene glycol treatment.

Brief (approximately 20-min) exposure of S49 lymphoma cells to beta-agonists such as isoproterenol leads to a homologous form of desensitization in which beta-agonist but not prostaglandin E1-sensitive or NaF-sensitive adenylate cyclase is reduced. The desensitized receptors (R) appear to be sequestered away from the effector system (guanine nucleotide regulatory protein (Ns) and adenylate cyclase (C)). Membrane perturbants such as polyethylene glycol are known to reorient membrane proteins and lipids. Thus, we fused agonist-desensitized S49 lymphoma cells to each other, using polyethylene glycol as fusogen, in an attempt to functionally reunite the R, N, and C components which might have become sequestered in microdomains of the plasma membrane during desensitization. Such treatment completely restored isoproterenol-stimulated adenylate cyclase to normal and re-established the ability of R and N to functionally couple as assessed by the ability to form a high affinity, guanine nucleotide-sensitive state of the receptor. These results support the concept that agonist-promoted sequestration plays a functionally significant role in the homologous desensitization of the beta-adrenergic receptor.

Adenylyl Cyclases↗
PubMed1985

Homologous desensitization of adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor.

We recently demonstrated that heterologous desensitization of adenylate cyclase in turkey erythrocytes is highly correlated with phosphorylation of the beta-adrenergic receptor. In contrast, little is known of the biochemical mechanisms underlying the homologous form of beta-adrenergic receptor desensitization, which is agonist-specific and not cAMP-mediated. Accordingly, the present studies were undertaken to examine if phosphorylation of the beta-adrenergic receptor is also associated with this form of desensitization in a well studied model system, the frog erythrocyte. Preincubation of these cells with the beta-adrenergic agonist isoproterenol leads to a 45% decline in isoproterenol-stimulated adenylate cyclase activity without significant changes in basal, prostaglandin E1-, NaF-, guanyl-5'-yl-imidodiphosphate-, forskolin-, or MnCl2-stimulated enzyme activities. There is also a 48% decline in [125I]iodocyanopindolol membrane binding sites. Conversely, preincubation of the cells with prostaglandin E1 attenuates only the prostaglandin E1-stimulated enzyme activity and does not affect [125I]iodocyanopindolol binding. Phosphorylation of the beta-adrenergic receptor was assessed by preincubating the cells with 32Pi and desensitizing them, and subsequently purifying the receptors by affinity chromatography. Under basal conditions there is about 0.62 mol of phosphate/mol of receptor whereas after desensitization with isoproterenol this increases to 1.9 mol/mol. This isoproterenol-induced receptor phosphorylation exhibits stereospecificity and is blocked by the beta-adrenergic antagonist propranolol. In addition, preincubation with prostaglandin E1 does not promote beta-adrenergic receptor phosphorylation. These data suggest that receptor phosphorylation is involved in homologous as well as heterologous forms of desensitization and may provide a unifying mechanism for desensitization of adenylate cyclase-coupled hormone receptors.

Adenylyl Cyclases↗
PubMed1985

Desensitization of the turkey erythrocyte beta-adrenergic receptor in a cell-free system. Evidence that multiple protein kinases can phosphorylate and desensitize the receptor.

We have used a recently developed cell-free system (cell lysate) derived from turkey erythrocytes to explore the potential role of cAMP-activated and other protein kinase systems in desensitizing the adenylate cyclase-coupled beta-adrenergic receptor. Desensitization by the agonist isoproterenol required more than simple occupancy of the receptor by the agonist since under conditions where adenylate cyclase was not activated, no desensitization occurred. As in whole cells, addition of cyclic nucleotides to the cell lysate produced only approximately 50% of the maximal isoproterenol-induced desensitization obtainable. Addition of the purified cAMP-dependent protein kinase holoenzyme plus isoproterenol to isolated turkey erythrocyte plasma membranes mimicked the submaximal desensitization induced in lysates by cAMP. This effect was entirely blocked by the specific inhibitor of the cAMP-dependent protein kinase. By contrast, maximal desensitization induced in lysates by isoproterenol was only approximately 50% attenuated by the protein kinase inhibitor. In the lysate preparations, isoproterenol was also shown to induce, in a stereospecific fashion, phosphorylation of the beta-adrenergic receptor. Phosphorylation promoted by isoproterenol was attenuated by cAMP-dependent protein kinase inhibitor to the same extent as desensitization (i.e. approximately 50%). Phorbol diesters also promoted receptor desensitization and phosphorylation in cell lysates. The desensitization was mimicked by incubation of isolated turkey erythrocyte membranes with partially purified preparations of protein kinase C plus phorbol diesters. In the cell lysate, calmodulin also promoted receptor phosphorylation and desensitization which was blocked by EGTA. Desensitization of adenylate cyclase by isoproterenol, phorbol diesters, and calmodulin was not observed to be additive. These findings suggest that: (a) multiple protein kinase systems, including cAMP-dependent, protein kinase C-dependent, and Ca2+/calmodulin-dependent kinases, are capable of regulating beta-adrenergic receptor function via phosphorylation reactions and that (b) cAMP may not be the sole mediator of isoproterenol-induced phosphorylation and desensitization in these cells.

Adenylyl Cyclases↗
PubMed1985

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles.

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.

Adenylyl Cyclases↗
PubMed1985

Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.

DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of alpha 1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular alpha 1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same rapid time course with a t1/2 less than 2 min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.

Animals↗
PubMed1985

The impact of aging on adrenergic receptor function: clinical and biochemical aspects.

From this discussion, several conclusions can be drawn. First, with advancing age there is a decrease in cardiovascular responsiveness and, more specifically, there is a decrease in catecholamine-stimulated chronotropic and inotropic responses. This decreased function has its biochemical correlate in the observation that cyclic AMP levels are decreased in response to isoproterenol infusion in cells or tissues derived from aged organisms. Second, although most work on human circulating cells suggests that beta-adrenergic receptor densities are unchanged, measurements of beta-adrenergic receptor concentrations in various cells from various animals (predominantly rats) have yielded conflicting results. Some of this disparity could be due to the observation that local concentrations of norepinephrine, such as those found intramyocardially, may be very different from those in circulating plasma. Indeed, whereas circulating norepinephrine levels tend to rise with age, the intramyocardial norepinephrine levels tend to fall with senescence. Thus, circulating lymphocytes may or may not be an appropriate model to reflect the catecholamine milieu to which other tissues may be exposed. Accordingly, a note of caution must be entered in terms of extrapolating findings regarding the levels of human lymphocyte beta-adrenergic receptors and cyclic AMP activity to those found, for example, in the human heart. Furthermore, it is likely that age-related changes in adrenergic function may be the result of changes in coupling of receptors to the adenylate cyclase system, as suggested by Feldman and co-workers, and/or changes in steps distal to cyclase activation, as suggested by Guarnieri and colleagues.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1985

Sensitization of frog erythrocyte adenylate cyclase system by tumor-promoting phorbol diesters.

Preincubation of frog erythrocyte lysates with tumor-promoting phorbol diesters leads to an increase in adenylate cyclase activity. This stimulatory effect of phorbol diesters was specific. Incubation with 12-O-tetradecanoylphorbol 13-acetate led to increases in basal (38%) and isoproterenol- (40%), fluoride- (25%), and Mn-stimulated (68%) adenylate cyclase activities compared with control. The inactive phorbol diesters (4 alpha-phorbol 12,13-didecanoate and beta-phorbol) were ineffective in promoting increases in adenylate cyclase activity. The effect of active phorbol diesters was also observed on isolated frog erythrocyte membranes in the absence of cell supernatant, although to a much lesser extent than in the whole lysates. Addition of the cell supernatant or of purified protein kinase C to the membranes maximized the sensitization by the phorbol diesters. These data are consistent with the notion that some component(s) of the adenylate cyclase system is (are) phosphorylated by protein kinase C, resulting in an enhancement of enzyme activity.

Adenylyl Cyclases↗
PubMed1985

Covalent labeling of the beta-adrenergic ligand-binding site with para-(bromoacetamidyl)benzylcarazolol. A highly potent beta-adrenergic affinity label.

para-(Bromoacetamidyl)benzylcarazolol (pBABC) was synthesized and found to be an extremely potent affinity label for beta-adrenergic receptors. Its interaction with mammalian (rabbit and hamster lung) and nonmammalian (turkey and frog erythrocyte) beta-adrenergic receptors was similar, displaying EC50 values of 400-900 pM for inhibiting 125I-cyanopindolol binding to these receptors. pBABC reduced the number of beta-adrenergic receptors in frog erythrocyte membranes, without any change in the affinity of the remaining sites for [125I]iodocyanopindolol. pBABC has been radioiodinated. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this affinity probe specifically labeled the beta-adrenergic peptide of a purified preparation of hamster lung, with high efficiency (approximately 40%) and with a pharmacological specificity characteristic of an interaction at the beta 2-adrenergic receptor ligand-binding site. Comparison of the proteolyzed products derived from purified receptor labeled with [125I]pBABC and with the photoaffinity agent [125I]p-azidobenzylcarazolol suggested that covalent labeling of the beta-adrenergic receptor by these probes occurs at similar domains of the beta-adrenergic receptor. Because of the much higher level of incorporation of this affinity probe as opposed to photosensitive probes, pBABC should prove to be a useful tool for structural studies of purified beta-adrenergic receptors.

Affinity Labels↗
PubMed1985

Homologous desensitization of the beta-adrenergic receptor. Functional integrity of the desensitized receptor from mammalian lung.

Previous work has demonstrated that injection of rats with isoproterenol is rapidly (10 min) followed by the development of a homologous form of desensitization of the beta-agonist-coupled adenylate cyclase in lung membranes. Half the receptor pool becomes sequestered in a light membrane fraction while the other half remains in the plasma membranes but becomes functionally uncoupled. In the present work we sought to assess whether "local sequestration" of the functionally intact receptor away from the effector adenylate cyclase in the plasma membrane contributes to the uncoupling of the beta-adrenergic receptor observed in the plasma membranes. We tested the functionality of the desensitized beta-adrenergic receptor in three different ways. We reconstituted the affinity chromatography purified control and "desensitized" receptors with pure Ns from human erythrocytes and assessed the ability to induce GTPase activity in Ns. Both control and desensitized beta-adrenergic receptors stimulate similar levels of GTPase activity in Ns (852 +/- 38 versus 738 +/- 49 fmol of Pi released/30 min (p greater than 0.05, n = 4). To further assess the relative ability of control and desensitized beta-adrenergic receptors to couple to another source of Ns we fused reconstituted beta-adrenergic receptors to Xenopus laevis erythrocytes, which contain Ns and adenylate cyclase but essentially no beta-adrenergic receptors. The functional interactions of control and desensitized beta-adrenergic receptor with the adenylate cyclase system of the acceptor cells was assessed by measuring the beta-agonist-stimulated adenylate cyclase activity and the agonist-induced formation of the high affinity state of the beta-adrenergic receptor (RH). Again both control and desensitized beta-adrenergic receptors appeared to interact with Ns to the same extent. To test if a local sequestration of the beta-adrenergic receptor away from Ns within the plasma membrane might contribute to the uncoupling of the beta-adrenergic receptors during desensitization, plasma membranes from control and desensitized lungs were treated with the fusogen polyethylene glycol to disrupt any compartmentalization of protein components within the plasma membrane. After polyethylene glycol treatment the previously uncoupled beta-adrenergic receptors could be recoupled to Ns as assessed by the formation of RH in agonist competition curves. These data suggest that in marked contrast to the heterologous type of desensitization, homologous desensitization may involve a local sequestration of a functionally intact beta-adrenergic receptor away from the adenylate cyclase effector system.

Adenylyl Cyclases↗
PubMed1984

Endogenous proteinases modulate the function of the beta-adrenergic receptor-adenylate cyclase system.

Photoaffinity labeling techniques have recently demonstrated that mammalian beta 1- and beta 2-adrenergic receptors reside on peptides of Mr 62 000-64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of Mr 30 000-55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the beta 2-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of Mr 40 000-55 000 are fully functional with respect to their ability to bind beta-adrenergic antagonist radioligands such as [3H]dihydroalprenolol and beta-adrenergic antagonist photoaffinity reagents such as p-azido-m-[125I]iodobenzylcarazolol. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc- variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of beta-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.

Adenylyl Cyclases↗
PubMed1984

Enhanced adenylate cyclase activity of turkey erythrocytes following treatment with beta-adrenergic receptor antagonists.

The turkey erythrocyte contains a beta 1-adrenergic receptor-linked adenylate cyclase system. We have examined the effects of pretreatment with receptor antagonists on adenylate cyclase activity and the individual components in the pathway of enzyme activation in this system. Isoproterenol-stimulated adenylate cyclase activity is increased by 30% (P less than 0.01) over control in membranes derived from cells preincubated with the antagonist propranolol. The effect is stereospecific and dose-related with a EC50 of 100 nM for the (-) isomer. The time course of effect is rapid being complete by 90 min. Non-receptor mediated stimulation of adenylate cyclase activity by manganese ion, forskolin and NaF is similarly enhanced following propranolol pretreatment. Sensitization of adenylate cyclase activity also occurs following pretreatment with a number of antagonists but is not seen after preincubation with pindolol or practolol. Quantitation of beta-adrenergic receptor (R) density using [125I]cyanopindolol indicates no difference between membranes derived from control and antagonist pretreated cells. Coupling of R with the guanine nucleotide regulatory protein (N) as assessed by high affinity agonist binding is unchanged following pretreatment. The efficacy of 5'-guanylylimidodiphosphate Gpp(NH)p in producing a shift of agonist binding curves associated with destabilization of high affinity H-R-N complexes, is also the same (EC50 = 0.2 microM) in membranes from control and antagonist treated cells. The isoproterenol stimulated rate of release of [3H]GDP from membranes preloaded with [3H]GTP as an index of formation of an active form of the N protein is similarly unaffected by antagonist preincubation. We conclude that the mechanism of the observed sensitization of turkey erythrocyte adenylate cyclase by beta-adrenergic antagonists is receptor mediated and likely involves facilitation of N interaction with the catalytic subunit of the enzyme.

Adenylyl Cyclases↗
PubMed1984

Photoaffinity labeling of beta-adrenergic receptors in mammalian tissues.

Photoaffinity labeling of beta 1- and beta 2-adrenergic receptors in plasma membranes from various mammalian tissues has been been performed utilizing the recently developed beta-adrenergic antagonist probe [125I]para-azidobenzylcarazolol. Tissues studied and their proportions of beta 1 and beta 2 receptors were: rat lung (18% beta 1, 82% beta 2), rabbit lung (72% beta 1, 28% beta 2), guinea pig lung (15% beta 1, 85% beta 2), dog lung (20% beta 1, 80% beta 2) and rabbit skeletal muscle (10% beta 1, 90% beta 2). As assessed by autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two to three specifically protected bands of Mr 62,000-65,000, 50,000-55,000 and 38,000-42,000 were observed in each tissue system. In each case, beta-adrenergic agonists and antagonists protected against photolabeling with appropriate beta 1 and beta 2 selectivity. Thus, in rat lung the beta 2 selective antagonist ICI-118,551 was more potent in blocking incorporation than the beta 1 selective antagonist betaxolol, whereas in rat, dog and guinea pig lung and rabbit skeletal muscle epinephrine was more potent than norepinephrine in blocking labeling, indicating a beta 2 specificity in these tissues. Conversely, in rabbit lung membranes, norepinephrine was approximately equipotent with epinephrine in blocking photoincorporation, indicating a beta 1 selectivity. In some systems protease inhibitors, especially those specific for metalloproteases (EDTA, EGTA), markedly diminished the amount of the smaller Mr peptides. For example, in rat lung the ratio of Mr 62,000:47,000:36,000 peptides changed from 30:40:30 to 60:35:5 in the presence of inhibitors. These results demonstrate the applicability of using [125I]para-azidobenzylcarazolol to covalently label mammalian beta-adrenergic receptors and suggest that mammalian beta 1 and beta 2 receptor binding sites primarily reside on peptides of Mr 62,000-65,000 and that smaller ligand binding fragments may arise by proteolysis.

Affinity Labels↗
PubMed1984

Association of sequestered beta-adrenergic receptors with the plasma membrane: a novel mechanism for receptor down regulation.

Chronic exposure of frog erythrocytes to beta-adrenergic agonists leads to desensitization of the responsiveness of adenylate cyclase to isoproterenol and is accompanied by "down-regulation", a decrease in the number of beta-adrenergic receptors on the cell surface. When frog erythrocyte plasma membranes are prepared by osmotic lysis of cells, the receptors lost from the cell surface during desensitization can be recovered in a "light membrane fraction", obtained by centrifuging the cell cytosol at 158,000 X g for 1 hr. These receptors are sequestered away from the plasma membrane fraction which contains the adenylate cyclase and the guanine nucleotide regulatory protein. If desensitized frog erythrocytes are disrupted by gentler freeze/thaw procedures, however, the sequestered beta-adrenergic receptors can be demonstrated to be physically associated with the plasma membrane. Typically, plasma membranes prepared in this fashion do not demonstrate a significant down regulation despite attenuation of isoproterenol-stimulated adenylate cyclase activity. Under these conditions, beta-adrenergic receptors from control and desensitized preparations co-migrate on sucrose density gradients in exactly the same place as the plasma membrane marker, adenylate cyclase. In contrast, when membranes from osmotically lysed desensitized cells are fractionated on sucrose gradients the down regulated receptors are sequestered in a light membrane fraction which barely enters the gradient and which is physically separated from adenylate cyclase activity. The data are consistent with a novel mechanism of receptor down-regulation which appears to involve the sequestration of the beta-adrenergic receptors away from the cell surface into a membrane compartment which remains physically associated with the plasma membrane.

Adenylyl Cyclases↗
PubMed1984

The mammalian beta 2-adrenergic receptor: purification and characterization.

The beta 2-adrenergic receptors from hamster, guinea pig, and rat lungs have been solubilized with digitonin and purified by sequential Sepharose-alprenolol affinity and high-performance steric-exclusion liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveal a peptide with an apparent Mr of 64 000 in all three systems that coincides with the peptide labeled by the specific beta-adrenergic photoaffinity probe (p-azido-m-[125I]iodobenzyl)carazolol. A single polypeptide was observed in all three systems, suggesting that lower molecular weight peptides identified previously by affinity labeling or purification in mammalian systems may represent proteolyzed forms of the receptor. Purification of the beta-adrenergic receptor has also been assessed by silver staining, iodinated lectin binding, and measurement of the specific activity (approximately 15 000 pmol of [3H]dihydroalprenolol bound/mg of protein). Overall yields approximate 10% of the initial crude particulate binding, with 1-3 pmol of purified receptor obtained/g of tissue. The purified receptor preparations bind agonist and antagonist ligands with the expected beta 2-adrenergic specificity and stereoselectivity. Peptide mapping and lectin binding studies of the hamster, guinea pig, and rat lung beta 2-adrenergic receptors reveal significant similarities suggestive of evolutionary homology.

Animals↗