Showing papers by "Robert J. Lefkowitz published in 1986"
TL;DR: Cloning of the gene and cDNA for the mammalian β2AR indicates significant amino-acid homology with bovine rhodopin and suggests that, like rhodopsin7, βAR possesses multiple membrane-spanning regions.
Abstract: The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.
1,225 citations
TL;DR: 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 agonists-specific form of adenylate cyclase desensitization and suggests a general mechanism for regulation of receptor function.
Abstract: 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.
625 citations
TL;DR: In this paper, it was shown 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.
Abstract: 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.
218 citations
TL;DR: It is reported here that βARK is also capable of phosphorylating rhodopsin in a totally light-dependent fashion, and rhodopin kinase can phosphorylate the agonist-occupied βAR.
Abstract: The structural components involved in transduction of extracellular signals as diverse as a photon of light impinging on the retina or a hormone molecule impinging on a cell have been highly conserved. These components include a recognition unit or receptor (for example, the β-adrenergic receptor (βAR) for catecholamines or the ‘light receptor’ rhodopsin), a guanine nucleotide regulatory or transducing protein, and an effector enzyme (for example, adenylate cyclase or cyclic GMP phosphodiesterase)1,2. Molecular cloning has revealed that the βAR shares significant sequence and three-dimensional homology with rhodopsin3. The function of the βAR is diminished by exposure to stimulatory agonists, leading to desensitization4. Similarly, ‘light adaptation’ involves decreased coupling of photoactivated rhodopsin to cGMP phosphodiesterase activation5–7. Both forms of desensitization involve receptor phosphorylation. The latter is mediated by a unique protein kinase, rhodopsin kinase, which phosphorylates only the light-bleached form of rhodopsin8–10. An analogous enzyme (termed βAR kinase or βARK) phosphorylates only the agonist-occupied βAR11. We report here that βARK is also capable of phosphorylating rhodopsin in a totally light-dependent fashion. Moreover, rhodopsin kinase can phosphorylate the agonist-occupied βAR. Thus the mechanisms which regulate the function of these disparate signalling systems also appear to be similar.
188 citations
TL;DR: The successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles suggests 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.
185 citations
TL;DR: This review has focused on the regulatory role of receptor phosphorylation, and it is by no means the authors' intent to suggest that receptors are the only locus for physiological control of sensitivity to hormone and drug reaction.
Abstract: 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.
158 citations
TL;DR: The 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.
Abstract: 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.
144 citations
TL;DR: 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.
Abstract: 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.
123 citations
TL;DR: Exposure of the purified receptor to the sulfhydryl-specific reagent, phenylmercuric chloride, resulted in an 80% loss of binding activity, but no common partial proteolytic products were found in a comparison with purified beta 2-adrenergic receptors.
89 citations
TL;DR: The specific activity of purified receptor preparations is consistent with that expected for a pure peptide of Mr = 80,000 containing a single ligand binding site, and overall yields approximate 14% of initial crude particulate binding.
78 citations
TL;DR: Pretreatment of intact frog erythrocytes with TPA produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E1-, forskolin-, NaF-, and MnCl2-stimulated adenylate cyclase activities in frog ERYthrocyte membranes.
TL;DR: The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns during the activation of the regulatory protein and the stimulation of adenylate cyclase.
TL;DR: Recent insights into homologies between the receptor components of these two systems that have emerged from the molecular cloning of the β-adrenergic receptors and the discovery of the novel enzyme β- adrenergic receptor kinase are reviewed.
TL;DR: The α1‐ and β1‐adrenergic receptors were covalently labeled with the specific photoaffinity probes 4‐amino‐6,7‐dimethoxy‐2‐{4‐[5‐(4‐azido‐3‐[125I]iodo‐phenyl) pentanoyl]‐1‐piperazinyl}quinazoline (125I‐APDQ) and 125I‐p‐azidobenzylcaraz
Abstract: The adrenergic receptors of rat pineal gland were investigated using radiolabeled ligand binding and photoaffinity labeling techniques. 125I‐2‐[β‐(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 α1‐adrenergic receptors. 125I‐CYP‐labeled β‐adrenergic receptors were characterized as a homogeneous population of β1‐adrenergic receptors. The α1‐ and β1‐adrenergic receptors were covalently labeled with the specific photoaffinity probes 4‐amino‐6,7‐dimethoxy‐2‐{4‐[5‐(4‐azido‐3‐[125I]iodo‐phenyl) pentanoyl]‐1‐piperazinyl}quinazoline (125I‐APDQ) and 125I‐p‐azidobenzylcarazolol (125I‐pABC). 125I‐APDQ labeled an α1‐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 β1‐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. (Less)
TL;DR: Results indicate that tyrosine residues have a function in the conformational stability of α2-adrenoceptors and may be directly involved with ligand binding to the receptor.
TL;DR: Following photolysis up to 90% of the ..cap alpha../sub 2/-adrenoceptors could be irreversibly labeled with (/sup 3/H)SKF 102229.
Journal Article•
01 Jan 1986
TL;DR: This chapter discusses the progress that has been made in the purification of various components of the adenylate cyclase system and presents various types of reconstitution approaches that have been used to monitor the interactions of the β -adrenergic receptor with the other components of this system.
Abstract: Publisher Summary This chapter discusses the progress that has been made in the purification of various components of the adenylate cyclase system. In addition, it presents various types of reconstitution approaches that have been used to monitor the interactions of the β -adrenergic receptor with the other components of the adenylate cyclase system. The development of these approaches has proceeded in a systematic fashion from the rather simple two-component systems—containing the pure receptor and pure nucleotide regulatory protein—to the more complex systems containing both stimulatory and inhibitory components. These systems, for the first time, have provided a way to assess the biological activities of various components of the adenylate cyclase system. Regarding the β -adrenergic receptor, researchers have been able to ascertain that the peptide isolated by affinity chromatography carries both ligand-binding site and activating function of the receptor.
05 Mar 1986
TL;DR: Phosphorylation of ..beta..AR in the plasma membrane promotes their translocation away from themore » cell surface into a sequestered membrane domain where the phosphorylation is reversed, thus, enabling the return of .. beta..AR back to the cell surface and recoupling with AC.
Abstract: The authors utilized the frog erythrocyte (FE) as a ..beta..-adreneric receptor (..beta..AR) model system in which to study homologous desensitization. Preincubation with isoproterenol (ISO) leads to a 50% decline in ISO-stimulated adenylate cyclase (AC) activity without significant changes in basal, PGE/sub 1/-, NaF-, GppNHp-, forskolin-, or MnCl/sub 2/-stimulated AC activities. ISO treatment also induces the sequestration of ..beta..AR from the cell surface as evidenced by a 35% decline in (/sup 3/H)CGP-12177 binding sites on the surface of intact FE. Treatment of intact FE with ISO also promotes ..beta..AR phosphorylation to 2 mol PO/sub 4//mol of ..beta..AR. At 25/sup 0/C, the time courses of ISO-induced AC desensitization, ..beta..AR sequestration and ..beta..AR phosphorylation are identical occurring without a lag and exhibiting a t 1/2 of 30 min and a maximal response at 2.5 hrs. The sequestered ..beta..AR can be partially recovered upon cell lysis in a light membrane fraction (LMF), separable from the plasma membranes using sucrose gradients or differential centrifugation. ..beta..AR phosphorylation is reversed in the sequestered LMF exhibiting a PO/sub 4//..beta..AR stoichiometry of 0.7 mol/mol - similar to that observed under basal conditions. These data suggest that phosphorylation of ..beta..AR in the plasma membrane promotes their translocation away from themore » cell surface into a sequestered membrane domain where the phosphorylation is reversed, thus, enabling the return of ..beta..AR back to the cell surface and recoupling with AC.« less