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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 325 records · Page 18Linked to original sources
PubMed1987

The multiple membrane spanning topography of the beta 2-adrenergic receptor. Localization of the sites of binding, glycosylation, and regulatory phosphorylation by limited proteolysis.

The beta 2-adrenergic receptor (beta-AR) is an integral membrane glycoprotein of apparent Mr approximately equal to 64,000. The amino acid sequence deduced from the beta-AR gene reveals homology with the visual pigment rhodopsin of retinal rod outer segments. We have proposed a structural model of beta-AR which is similar to that elucidated for rhodopsin. In this paper we identify a number of structural and topographical characteristics of beta-AR consistent with the model through the use of limited proteolysis. Limited trypsinization of beta-AR reconstituted in lipid vesicles yields two insoluble (integral membrane) domains of Mr approximately equal to 38,000 and 26,000. Identical results were obtained in intact cells, indicating that the cleavage site of the receptor is accessible at the extracellular surface of the plasma membrane. The amino-terminal domain (38 kDa) contains the ligand binding site (as revealed by photoaffinity labeling) and the sites of glycosylation (as revealed by its sensitivity to endoglycosidase F), whereas the carboxyl-terminal domain (26 kDa) contains all the sites of in vitro phosphorylation by cAMP-dependent protein kinase and the beta-adrenergic receptor kinase. Of four canonical sites for N-linked glycosylation, two near the amino and two near the carboxyl terminus, only those in the amino-terminal domain (Asn6 and Asn15) are utilized and sensitive to endoglycosidase F. Carboxypeptidase Y treatment of reconstituted native beta-adrenergic receptor generates a truncated (approximately 57 kDa) glycopeptide that has lost most of the sites phosphorylated by beta-AR kinase and one of the sites phosphorylated by protein kinase A. The various features delineated, including the length of the carboxypeptidase Y-sensitive region, the extracellular location of the trypsin-sensitive site, the location of the sites of phosphorylation and glycosylation all constrain the receptor to a rhodopsin-like structure with multiple membrane spanning segments.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of the beta-adrenergic receptor kinase.

The beta-adrenergic receptor kinase (beta-ARK) is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta-AR) as well as the light-bleached form of rhodopsin. beta-ARK is present in a wide variety of mammalian tissues. The kinase can be purified from bovine cerebral cortex to greater than 90% homogeneity by sequential chromatography on Ultrogel AcA34, DEAE-Sephacel, CM-Fractogel, and hydroxylapatite. This results in an approximately 20,000-fold purification with an overall recovery of 12%. The purified kinase has an Mr approximately 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several findings indicate that this peptide contains the beta-ARK activity. First, on hydroxylapatite chromatography the enzyme activity coelutes with the Mr approximately 80,000 protein as revealed by Coomassie-Blue staining. Second, under phosphorylating conditions the Mr approximately 80,000 protein is phosphorylated. Finally, the Mr approximately 80,000 protein specifically interacts with reconstituted agonist-occupied beta-AR. Kinetic parameters of the enzyme for beta-AR are Km = 0.25 microM and Vmax = 78 nmol/min/mg whereas for rhodopsin the values are Km = 6 microM and Vmax = 72 nmol/min/mg. The Km value of the enzyme for ATP is approximately 35 microM using either beta-AR or rhodopsin as substrate. Receptor phosphorylation by beta-ARK is effectively inhibited by Zn2+, digitonin and a variety of salts. The availability of purified beta-ARK should greatly facilitate studies of its role in receptor desensitization.

Animals↗
PubMed1987

Delineation of the intronless nature of the genes for the human and hamster beta 2-adrenergic receptor and their putative promoter regions.

The beta 2-adrenergic receptor is the first adenylate cyclase-coupled receptor to be cloned. We provide here a detailed characterization of its complete gene in both the human and hamster which reveals several unusual and provocative features. The genes are present in a single copy, are intronless, and are bounded by homologous 18-bp (base pair) direct repeats. These findings suggest that the beta 2-adrenergic receptor may have arisen as a processed gene for another related gene. Genomic Southern blots done at reduced stringency in fact reveal additional weak signals. The human and hamster gene sequences 5' to the principal site of transcription initiation are highly homologous and share many characteristics of promoters for housekeeping genes. Moreover, there is present in the human genome a long (777 bp) open reading frame which is in frame with the beta-adrenergic receptor coding block and which ends only 234 bp 5' to the initiator methionine of the receptor. An unusual cDNA has been found, transcribed from a putative second more 5' promoter which contains the 5' half of the beta-adrenergic receptor as well as 1065-bp 5' to the receptor coding region, including the entire upstream long open reading frame (sufficient to encode a putative protein of Mr approximately 28,000).

Animals↗
PubMed1987

Somatostatin induces translocation of the beta-adrenergic receptor kinase and desensitizes somatostatin receptors in S49 lymphoma cells.

The beta-adrenergic receptor kinase is a cytosolic enzyme that specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta AR). Beta AR kinase appears to be translocated from the cytosol to the plasma membrane when kin- S49 lymphoma cells are incubated with either beta-adrenergic agonists or prostaglandin E1, both of which act through receptors which stimulate adenylate cyclase. We report here that brief (approximately 20 min) exposure of wild type S49 lymphoma cells to somatostatin (which inhibits adenylate cyclase) promotes the translocation of beta AR kinase to an extent comparable to that observed in the presence of the beta agonist isoproterenol or prostaglandin E1. Beta AR kinase activity can be measured using either beta AR or rhodopsin, the retinal receptor for light, as a substrate. The translocation process triggered by somatostatin is rapid, reversible, and is associated with somatostatin receptor desensitization. The latter is apparent as an attenuation of the inhibition by somatostatin of forskolin-stimulated adenylate cyclase activity in membranes of S49 cells preincubated in the presence of the peptide. These results strongly suggest that beta AR kinase is able to phosphorylate and desensitize both stimulatory and inhibitory adenylate cyclase-coupled receptors, thus emerging as a general kinase that regulates the function of different receptors in an agonist-specific fashion.

Animals↗
PubMed1987

Regulation of transmembrane signaling by receptor phosphorylation.

At least two major effects of receptor phosphorylation have been identified--regulation of receptor function, and regulation of receptor distribution. In many cases where phosphorylation directly alters the functions of receptors, this appears to be in a negative direction. Such decreases in receptor activity may reflect reduced ability to interact with biochemical effectors (e.g., the beta-adrenergic receptor, rhodopsin), reduced affinity for binding agonist ligands (EGF,IGF-I, insulin receptors) or reduced enzymatic activity (e.g., tyrosine kinase activity of the insulin or EGF receptor). In all instances, these negative modulations are associated with phosphorylation of serine and/or threonine residues of the receptor proteins. In contrast, the tyrosine kinase receptors also appear to be susceptible to positive modulation by phosphorylation. With these receptors, autophosphorylation of tyrosine residues may lead to enhanced protein-tyrosine kinase activity of the receptors and increased receptor function. In addition, the subcellular distribution of a receptor may be regulated by its phosphorylation status (e.g., the beta-adrenergic receptor, receptors for insulin, EGF, IGF-II, and transferrin). The emerging paradigm is that receptor phosphorylation may in some way promote receptor internalization into sequestered compartments where dephosphorylation occurs. The molecular and cellular mechanisms involved in translating changes in receptor phosphorylation into changes in receptor distribution remain to be elucidated. Moreover, the biological role of receptor internalization may be quite varied. Thus, in the case of the beta-adrenergic receptor, it may serve primarily as a mechanism for bringing the phosphorylated receptors into contact with intracellular phosphatases that dephosphorylate and resensitize it. By contrast, for the transferrin receptor and other receptors involved in receptor-mediated endocytosis, the internalization presumably functions to carry some specific ligand or metabolite into the cell. The role of phosphorylation in regulating receptor function dramatically extends the range of regulatory control of this important covalent modification.

Animals↗
PubMed1987

Functional differences in the beta gamma complexes of transducin and the inhibitory guanine nucleotide regulatory protein.

We have examined the mechanism of inhibition of adenylate cyclase using the purified alpha and beta gamma subunits of bovine brain inhibitory guanine nucleotide regulatory protein (Ni) (i.e., alpha i and beta gamma N) and bovine retinal transducin (alpha T and beta gamma T) in reconstituted phospholipid vesicle systems. The addition of beta gamma N or beta gamma T to lipid vesicles containing the pure stimulatory guanine nucleotide regulatory protein (Ns) from human erythrocytes as well as a resolved preparation of the catalytic moiety (C) of bovine caudate adenylate cyclase results in significant inhibition of guanine nucleotide stimulated cyclase activity (80-90%). The inhibition by these beta gamma subunit complexes appears to fully account for the inhibitory effects observed with holo-Ni or holotransducin. A variety of structure-function comparisons of the beta gamma N and beta gamma T complexes were performed in order to further probe the molecular mechanisms involved in the inhibitory pathway. Whereas the beta subunits of beta gamma N and beta gamma T appear to be very similar, if not identical, on the basis of comparisons of their gel electrophoretic mobility and immunological cross-reactivity, clear differences exist in the apparent structures of gamma N and gamma T. Interestingly, functional differences are observed in the effectiveness of these two beta gamma complexes to inhibit adenylate cyclase activity. Specifically, while both beta gamma N and beta gamma T are capable of effecting significant levels of inhibition of the guanine nucleotide stimulated activities, the beta gamma N complex is consistently more potent than beta gamma T in inhibiting these activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1987

Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of alpha 1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells.

Continuous exposure of DDT1 MF-2 smooth muscle cells to 10-100 microM norepinephrine results in a dramatic attenuation of the ability of norepinephrine to stimulate inositol phospholipid hydrolysis via alpha 1-adrenergic receptors (alpha 1-AR). In addition to the functional desensitization, norepinephrine exposure also reduces the number of accessible cell surface alpha 1-AR as assayed by [3H]prazosin binding at 4 degrees C. Desensitization of the cells with norepinephrine results in an increase in the phosphorylation of the Mr 80,000 alpha 1-AR ligand binding peptide (2.4 +/- 0.2 mol of 32P per mol of alpha 1-AR; n = 5) when compared to control cells (1.1 +/- 0.1 mol of 32P per mol of alpha 1-AR; n = 5). The time courses of these three processes are all comparable being half-maximal within 1-2 min. These norepinephrine-promoted effects can be prevented by the alpha 1-AR receptor antagonist phentolamine indicating that they are mediated via the alpha 1-AR. Treatment of cells with the vasoactive peptide bradykinin (10 microM) induces desensitization of alpha 1-AR function similar to that induced by tumor-promoting phorbol ester treatment (Leeb-Lundberg, L. M. F., Cotecchia, S., Lomasney, J. W., DeBernardis, J. F., Lefkowitz, R. J., and Caron, M. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5651-5655). Both treatments also result in phosphorylation of the alpha 1-AR, with stoichiometries of 1.7 +/- 0.1 (bradykinin; n = 5) and 3.6 +/- 0.1 (PMA; n = 5) mol of 32P/mol of alpha 1-AR. However, neither phorbol esters nor bradykinin reduce the number of accessible cell surface alpha 1-AR. Similar phosphopeptide maps are obtained from tryptic phosphopeptides generated from phosphorylated alpha 1-AR derived from cells treated with norepinephrine, phorbol 12-myristate 13-acetate, and bradykinin. Phosphoamino acid analysis reveals that the various agents induce phosphorylation on both serine and threonine residues. Thus, phosphorylation of receptors linked to the inositol phospholipid/Ca2+ signaling pathway may represent an important mechanism of regulation of receptor responsiveness.

Animals↗
PubMed1987

Regulation of adrenergic receptor function by phosphorylation. II. Effects of agonist occupancy on phosphorylation of alpha 1- and beta 2-adrenergic receptors by protein kinase C and the cyclic AMP-dependent protein kinase.

The present study was undertaken to determine the ability of protein kinase C and protein kinase A to directly phosphorylate the purified alpha 1- and beta 2-adrenergic receptors (AR). Both the catalytic subunit of protein kinase A and the protein kinase C, purified from bovine heart and pig brain, respectively, are able to phosphorylate the purified alpha 1-AR from DDT1 MF-2 smooth muscle cells. Occupancy of the receptor by an alpha 1 agonist, norepinephrine (100 microM), increases the rate of phosphorylation by protein kinase C but not by protein kinase A. The maximum stoichiometry of phosphorylation obtained is not affected by the agonist and reached 3 mol of PO4/mol of receptor for protein kinase C and 1 mol of PO4/mol of receptor for protein kinase A. The phosphopeptide maps of the trypsinized alpha 1-AR phosphorylated by each kinase differ drastically. The beta 2-AR purified from hamster lungs can also be phosphorylated by the two kinases. In contrast to the alpha 1-AR, the occupancy of the beta 2-AR by the agonist isoproterenol (20 microM) increases the rate of phosphorylation of the beta 2-AR by protein kinase A but not by protein kinase C. The maximum amount of phosphate incorporated into the receptor is not affected in either case by the agonist and reaches 1 mol of PO4/mol of receptor with protein kinase A and 0.4 mol of PO4/mol of receptor with protein kinase C. The phosphopeptide maps of the trypsinized receptor phosphorylated by either kinase reveal similar profiles. Thus, both alpha 1-AR and beta 2-AR are substrates for protein kinase A and protein kinase C. Agonist occupancy of the two receptors facilitates their phosphorylation only by the protein kinase coupled to their own signal transduction pathway. These observations suggest that "feedback" and "cross-system" phosphorylation may represent distinct and differently regulated mechanisms of modulation of receptor function.

Animals↗
PubMed1987

Molecular mechanisms of beta-adrenergic receptor desensitization.

Multiple mechanisms seem to be involved in regulating the responsiveness of hormone receptor-coupled adenylate cyclase systems. These mechanisms at least involve the receptors and nucleotide regulatory proteins. With the recent development of methods for purifying the catalytic unit of the enzyme it will be possible to assess whether it is also a locus for such regulatory phenomena. At least two major pathways of receptor regulation have been uncovered. Homologous desensitization (Fig. 9) involves the uncoupling and translocation of the receptors out of their normal plasma membrane environment. This process sequesters the receptors away from their effector, the regulatory and catalytic components of adenylate cyclase. The site of receptor sequestration is unclear and might lie within the plasma membrane or within the cell. The sequestered receptors can recycle to the cell surface or become down-regulated, perhaps being destroyed within the cell. Phosphorylation of the receptors through a beta-adrenergic receptor kinase appears to be associated with homologous desensitization. This phosphorylation event may serve either to uncouple functionally the receptors or to trigger their sequestration from the cell surface or both. In heterologous desensitization (Fig. 10), receptor function is regulated by phosphorylation in the absence of receptor sequestration or down-regulation. This covalent modification serves to functionally uncouple the receptors, that is, to impair their interactions with the guanine nucleotide regulatory proteins. Several protein kinases seem to be capable of promoting phosphorylation of the receptors including the cAMP-dependent kinase and protein kinase C. In addition to the receptor modification, heterologous desensitization seems to be associated with functional modifications (phosphorylation?) at the level of nucleotide regulatory proteins (Ns and Ni), (Fig. 10). Further studies of the mechanisms of desensitization of adenylate cyclase-coupled receptors are thus likely to help elucidate modes of regulation of a wide variety of receptor-coupled functions in diverse types of cells.

Adenylyl Cyclases↗
PubMed1987

Phosphorylation of the beta-adrenergic receptor in intact cells: relationship to heterologous and homologous mechanisms of adenylate cyclase desensitization.

We have recently shown that both heterologous and homologous forms of adenylate cyclase desensitization involve phosphorylation of beta-adrenergic receptors. In order to compare these two reactions, we wished to identify a single cell system in which both processes could be studied. Using the frog erythrocyte, which has been previously shown to exhibit cAMP-independent homologous desensitization, we have found that under appropriate conditions cAMP-dependent heterologous desensitization can be elicited. Incubation of intact cells with the membrane-permeable cAMP analogs dibutyryl cAMP or 8-bromo cAMP promotes about a 50% desensitization of isoproterenol- and prostaglandin E1-stimulated adenylate cyclase activity in a time-, temperature-, and dose-dependent fashion. There is also a 20% desensitization in the abilities of guanine nucleotides (GTP and guanyl-5'-yl-imidodiphosphate) and NaF to stimulate adenylate cyclase maximally. In contrast, there is no effect on forskolin- or MnCl2-stimulated enzyme activities. The desensitization response is specific for cAMP as dibutyryl cGMP, 8-bromo cGMP, or 8-bromo AMP produce little or no desensitization. Incubation of the cells with dibutyryl cAMP does not affect the number of cell surface beta-adrenergic receptors. In contrast, incubation with isoproterenol promotes homologous desensitization and sequestration of the receptors. Incubation of 32P-labeled erythrocytes with either dibutyryl cAMP or isoproterenol promotes a stoichiometric threefold increase in the phosphorylation state of the beta-adrenergic receptor which occurs predominantly on serine residues. However, if the cells are coincubated with both dibutyryl cAMP and isoproterenol then the desensitization of isoproterenol-stimulated enzyme activity and phosphorylation of the beta-adrenergic receptor are greater than those observed with either agent alone. These results indicate that heterologous and homologous desensitization of adenylate cyclase-coupled beta-adrenergic receptors are mediated by different biochemical pathways involving phosphorylation of the receptor protein on distinct sites.

1-Methyl-3-isobutylxanthine↗
PubMed1987

cDNA for the human beta 2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor.

We have isolated and sequenced a cDNA encoding the human beta 2-adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster beta 2-adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. We have localized the gene for the beta 2-adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor.

Amino Acid Sequence↗
PubMed1987

Cloning of the cDNA for the human beta 1-adrenergic receptor.

Screening of a human placenta lambda gt11 library has led to the isolation of the cDNA for the human beta 1-adrenergic receptor (beta 1AR). Used as the probe was the human genomic clone termed G-21. This clone, which contains an intronless gene for a putative receptor, was previously isolated by virtue of its cross hybridization with the human beta 2-adrenergic receptor (beta 2AR). The 2.4-kilobase cDNA for the human beta 1AR encodes a protein of 477 amino acid residues that is 69% homologous with the avian beta AR but only 54% homologous with the human beta 2AR. This suggests that the avian gene encoding beta AR and the human gene encoding beta 1AR evolved from a common ancestral gene. RNA blot analysis indicates a message of 2.5 kilobases in rat tissues, with a pattern of tissue distribution consistent with beta 1AR binding. This pattern is quite distinct from the pattern obtained when the beta 2AR cDNA is used as a probe. Expression of receptor protein in Xenopus laevis oocytes conveys adenylate cyclase responsiveness to catecholamines with a typical beta 1AR specificity. This contrasts with the typical beta 2 subtype specificity observed when the human beta 2AR cDNA is expressed in this system. Mammalian beta 1AR and beta 2AR are thus products of distinct genes, both of which are apparently related to the putative G-21 receptor.

Amino Acid Sequence↗
PubMed1987

Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein).

The beta-adrenergic receptor kinase is an enzyme, possibly analogous to rhodopsin kinase, that multiply phosphorylates the beta-adrenergic receptor only when it is occupied by stimulatory agonists. Since this kinase may play an important role in mediating the process of homologous, or agonist-specific, desensitization, we investigated the functional consequences of receptor phosphorylation by the kinase and possible analogies with the mechanism of action of rhodopsin kinase. Pure hamster lung beta 2-adrenergic receptor, reconstituted in phospholipid vesicles, was assessed for its ability to mediate agonist-promoted stimulation of the GTPase activity of coreconstituted stimulatory guanine nucleotide-binding regulatory protein. When the receptor was phosphorylated by partially (approximately 350-fold) purified preparations of beta-adrenergic receptor kinase, as much as 80% inactivation of its functional activity was observed. However, the use of more highly purified enzyme preparations led to a dramatic decrease in the ability of phosphorylation to inactivate the receptor such that pure enzyme preparations (approximately 20,000-fold purified) caused only minimal (approximately 1off/- 7%) inactivation. Addition of pure retinal arrestin (48-kDa protein or S antigen), which is involved in enhancing the inactivating effect of rhodopsin phosphorylation by rhodopsin kinase, led to partial restoration of the functional effect of beta-adrenergic receptor kinase-promoted phosphorylation (41 +/- 3% inactivation). These results suggest the possibility that a protein analogous to retinal arrestin may exist in other tissues and function in concert with beta-adrenergic receptor kinase to regulate the activity of adenylate cyclase-coupled receptors.

Animals↗
PubMed1987

The mammalian beta-adrenergic receptor: structural and functional characterization of the carbohydrate moiety.

Mammalian beta-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of Mr approximately 64,000. Treatment of purified [125I]-labeled hamster lung beta-adrenergic receptor with alpha-mannosidase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [125I]-labeled receptor with endoglycosidase F results initially in the formation of a Mr approximately 57,000 peptide which is further converted to Mr approximately 49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [125I]-labeled receptor reveals the presence of two complex type carbohydrate chains (approximately 10% of which are fucosylated) on approximately 45% of the receptors. The remaining approximately 55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of beta-adrenergic agonists and antagonists. Moreover, the ability of control, alpha-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, alpha-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleotide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the beta-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.

Adrenergic beta-Agonists↗
PubMed1986

Chemical modification of alpha 2-adrenoceptors. Possible role for tyrosine in the ligand binding site.

Tetranitromethane (TNM) is a reagent which reacts with the tyrosine and cysteine residues of proteins. Chemical modification of partially purified human platelet alpha 2-adrenoceptors with TNM resulted in an irreversible loss of binding activity. Typically, an 80-90% decrease in binding activity occurred with a 60-min exposure to 320 microM TNM. The loss of alpha 2-adrenoceptor activity caused by TNM could be prevented if alpha 2-adrenergic ligands were present during exposure of the receptor to TNM. The protection afforded by alpha 2-adrenergic ligands was dose-dependent and showed a positive correlation with the affinity of the ligand for the alpha 2-adrenoceptor. Prazosin, an alpha 1-specific antagonist, and propranolol, a beta-adrenergic antagonist, did not protect alpha 2-adrenoceptors against the inactivation caused by TNM. Saturation curve analysis revealed that the decrease in alpha 2-adrenoceptor activity caused by TNM was due to a decrease in Bmax with no change in Kd. alpha 2-Adrenoceptors were also inactivated with the sulfhydryl-specific reagent phenylmercuric chloride (PMC). The receptor inactivation caused by PMC could be reversed completely by subsequent treatment with dithiothreitol. Treatment of alpha 2-adrenoceptors with combinations of TNM and PMC showed that the receptor inactivation caused by TNM was most likely due to an interaction with tyrosine residues. These results indicate that tyrosine residues have a function in the conformational stability of alpha 2-adrenoceptors and may be directly involved with ligand binding to the receptor.

Binding Sites↗