Showing papers by "Robert J. Lefkowitz published in 1982"

Reciprocal modulation of agonist and antagonist binding to muscarinic cholinergic receptor by guanine nucleotide.

Abstract: The ability of guanine nucleotide to decrease the binding affinity of agonists but not antagonists has been documented in a number of hormone and neurotransmitter receptor systems. By contrast, recent reports indicate that both agonist and antagonist binding to the muscarinic cholinergic receptors appear to be regulated in a reciprocal fashion by guanine nucleotide. We document two forms of the muscarinic cholinergic receptor in frog heart, which are present in approximately equal proportions and which display high-agonist/low antagonist and low-agonist/high-antagonist affinities, respectively. Guanine nucleotide appears to convert the former type of site into the latter type. These observations can be interpreted in terms of a model for two interconvertible forms of the muscarinic cholinergic receptor reciprocally favored by agonists and antagonists. This model has implications both for the understanding of neurotransmitter-receptor interactions generally and for the nature of the biological effects of receptor antagonists.

Interactions of agonists with platelet alpha 2-adrenergic receptors.

Abstract: Epinephrine induces human platelet aggregation by interacting with alpha-adrenergic receptors. These sites were demonstrated by radioligand-binding techniques using the new antagonist ligand, [3H]yohimbine. The sites labeled by [3H]yohimbine had the specificity of alpha 2-receptors with the affinity of yohimbine much greater than prazosin. Epinephrine-mediated inhibition of prostaglandin E1-stimulated adenylate cyclase activity in human platelet lysates was also found to have an alpha 2-receptor specificity. Competition curves of antagonists with [3H]yohimbine indicated a homogeneous population of alpha 2-receptors. In contrast, competition curves of a series of full and partial agonists with [3H]yohimbine were resolved into two distinct affinity states; the ratio of the dissociation constants of agonists for the low and high affinity states was positively correlated with the agonist's intrinsic activity for inhibition of adenylate cyclase. Guanine nucleotides were found to destabilize the high affinity form of the alpha 2-receptors. At high nucleotide concentrations, all high affinity states of the receptor were converted to the low affinity form. The formation of the high affinity agonist-binding state may reflect an interaction between the agonist-receptor complex and an additional membrane component, and probably reflects events involved in alpha 2-receptor-adenylate cyclase coupling.

The beta-adrenergic receptor: rapid purification and covalent labeling by photoaffinity crosslinking.

Abstract: New procedures for the rapid purification and covalent labeling of the beta-adrenergic receptors have been developed that should greatly accelerate progress in the study of these widely distributed adenylate cyclase-coupled receptors. Chromatography of solubilized receptor preparations on a Sepharose-alprenolol affinity gel followed by HPLC on steric exclusion columns lead to rapid (2 days) and high yield (approximately 30%) purification of the receptors from frog erythrocytes. The receptor obtained by these rapid procedures appears to be composed entirely of 58,000 Mr subunit(s) and to be identical to that previously purified by much lengthier procedures [Shorr, R. G. L., Lefkowitz, R. J. & Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826]. A novel, very high affinity, specific beta-adrenergic antagonist, p-aminobenzylcarazolol, has also been synthesized. It can be radioiodinated to theoretical specific radioactivity with 125I (2,200 Ci/mmol). This radioligand, which possesses an arylamine moiety, may then be covalently incorporated into the receptor binding subunit (58,000 Mr peptide) of the frog erythrocyte membranes by the use of the bifunctional photoactive crosslinker N-succinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate (SANAH). Covalent incorporation is blocked by various drugs with a strict beta-adrenergic specificity. This suggests that the photoaffinity crosslinking approach may be useful for labeling a variety of small molecule and neurotransmitter receptors when appropriate ligands can be synthesized.

Adrenergic Receptors in the Heart

Abstract: Introduction Catecholamines, acting through alphaand beta-adrenergic receptors, modulate a variety of physiological responses in the heart. Most impor­ tantly catecholamines increase the rate and force of cardiac contraction. These actions occur mainly as a consequence of the binding of the endoge­ nous substances norepinephrine and epinephrine to specific adrenergic receptors on the plasma membrane of cells in the heart. Whereas the effects of sympathetic nervous stimulation on the heart have been examined for many years (reviewed in 57), oIlly recently has it become possible to mea­ sure directly the properties of the receptors for catecholamines in the heart. Here we discuss a few selected areas of active research where radioligand binding techinques have been applied to the study of adrenergic receptors in the heart.

Mechanisms of hormone receptor-effector coupling: the beta-adrenergic receptor and adenylate cyclase.

Abstract: The beta-adrenergic receptors that are coupled to adenylate cyclase have provided a model system for studying the mechanisms by which a plasma membrane receptor is coupled to a well-defined biochemical effector. The beta 2-adrenergic receptors from frog erythrocyte membranes have been purified to homogeneity and the ligand-binding subunit has been identified as a glycoprotein with an approximate molecular weight of 58,000. This subunit has also been identified with the use of newly developed photoaffinity reagents. Under the influence of agonist hormones (H), the receptors (R) form transient complexes with another component of this system, termed the nucleotide regulatory protein (N). Formation of this ternary complex, HRN, leads to the dissociation of GDP from N and the interaction of stimulatory GTP with N. N charged with GTP appears to activate the catalytic moiety of the adenylate cyclase enzyme. Although some striking analogies have been found for the mechanisms by which inhibitory receptors interact with adenylate cyclase, much less is known about the molecular properties of the components involved and the ways in which they interact to dampen adenylate cyclase activity in the plasma membrane.

Clinical physiology of adrenergic receptor regulation

Abstract: Radioligand binding studies now permit the direct investigation of the alpha- and beta-adrenergic receptors. The concentration of the receptors in cell membranes is modulated by a wide variety of physiological and pathophysiological variables and may be either increased or decreased in different conditions. Ligand binding techniques have also shed light on the ways in which the adrenergic receptors are coupled to other components of the hormone-sensitive adenylate cyclase system. This “coupling” is also a highly regulated step, control of which importantly influences catecholamine and sensitivity of tissues.

Affinity chromatography of human platelet α2-adrenergic receptors

Abstract: Catecholamines, such as epinephrine, inhibit the enzyme adenylate cyclase (EC 4.6.1.1) via a specific receptor mechanism involving α2-adrenergic receptors. In order to facilitate purification of these inhibitory receptors we have prepared a highly effective biospecific affinity adsorbent. The immobilized ligand SKF 101253 is a 3-benzazepine with α2-adrenergic antagonist activity. SKF 101253 is coupled to Sepharose CL-4B by using a bifunctional reagent (1,4-butanediol diglycidyl ether) which also provides a hydrophilic spacer moiety between the ligand and the gel matrix. Membranes from human platelets, containing α2-adrenergic receptors, can be specifically labeled with [3H]yohimbine and can be solubilized with digitonin without loss of their α2-adrenergic binding characteristics. Chromatography of solubilized human platelet membrane preparations on the SKF 101253-Sepharose CL-4B affinity gel results in the adsorption of 70-80% of the initial [3H]yohimbine binding activity. Adsorption to the affinity gel is blocked by both α-adrenergic antagonists (phentolamine ≥ yohimbine > prazosin) and by α-adrenergic agonists [p-aminoclonidine > (-)-epinephrine > (+)-epinephrine]. Similarly, elution of specific [3H]yohimbine binding activity from the affinity gel is effected with the aforementioned agonists and antagonists in the same order of potency. Other drugs that do not interact appreciably with α-adrenergic receptors, such as (-)-isoproterenol, (-)-alprenolol, atropine, and carbachol, are ineffective for both the blockade of adsorption and the elution of specific [3H]yohimbine binding activity from the affinity gel. In addition to the specificity of the interaction, chromatography of solubilized human platelet membrane preparations on the SKF 101253-Sepharose CL-4B affinity gel results in a 40-50% overall yield and an approximately 200-fold increase in the specific binding activity for [3H]yohimbine. The results indicate that the SKF 101253-Sepharose CL-4B affinity adsorbent should provide a powerful tool for the purification of the adenylate cyclase-inhibitory α2-adrenergic receptor of human platelets.

Agonist interactions with alpha-adrenergic receptors.

Abstract: alpha-Adrenergic receptors have been grouped into two major subtypes, termed alpha 1- and alpha 2-receptors. Radioligand binding techniques have been utilized to measure the number of alpha 1- and alpha 2-receptors in a variety of tissues. [3H]Dihydroergocryptine labels the entire alpha-receptor population; the alpha-receptor subtypes may be delineated by constricting competition curves with unlabeled selective antagonists and analyzing the data with computer modeling techniques. Alternatively, alpha 1- and alpha 2-receptors may be directly identified with selective radioligands such as [3H]prazosin and [3H]yohimbine, respectively. For example, rat liver membranes have been shown to contain alpha 1- (80%) and alpha 2- (20%) receptors; the alpha 1-receptors activate glycogen phosphorylase. Radioligands have also been used to probe the mechanism by which alpha 2-receptors may inhibit adenylate cyclase activity. Agonist competition curves with [3H]dihydroergocryptine at eht human platelet's alpha 2-receptor may be resolved into two affinity components, interconvertible by guanine nucleotides. These data suggest the agonist-promoted association of the alpha 2-receptor with an additional membrane component. More direct evidence in favor of this possibility was indicated by the increase in sedimentation velocity of solubilized agonist-labeled receptor on sucrose density gradients.

Regulation of insulin receptors in frog erythrocytes by insulin and concanavalin A. Evidence for discrete classes of insulin binding sites.

Abstract: In order to better understand the complex nature of insulin-receptor interactions we have investigated insulin-promoted receptor down-regulation in frog and turkey erythrocytes. We have quantitatively analyzed the data for equilibrium insulin binding to frog erythrocytes before and after preincubation with insulin (down-regulation) or concanavalin A. Preincubation of frog erythrocytes with insulin for 4 hr at 30#{176} induced a 50% decrease in the ability of these cells to bind .125I-labeled insulin. Under identical experimental conditions, turkey erythrocytes exhibited no insulin-mediated insulin receptor down-regulation. The extent of down-regulation of the insulin receptor in frog erythrocytes was dependent upon the concentration of insulin (10_b0_10_6 M) and the time of exposure to hormone. However, down-regulation of the receptor was only slightly temperature-dependent and was not blocked at 4#{176} in these cells. When frog erythrocytes were incubated with 125I labeled insulin and the lysosomotropic amines chloroquine or methylamine, a 2-fold enhancement of cell-associated radioactivity was observed as compared with controls which received no chloroquine or methylamine. These findings suggest that the insulinreceptor complex in frog erythrocytes is internalized and degraded during the downregulation process. By contrast, turkey eyrthrocytes which do not down-regulate their receptors also appear not to internalize insulin. Detailed insulin binding isotherms for frog erythrocytes were subjected to nonlinear least-squares curve fitting. It was found that these curves could be adequately fit with a model for two classes of independent binding sites: Site I being of high affinity, low capacity (KH = 580 pM; R11 = 425 sites/cell) and Site II being of low affinity and high capacity (KL = 130 nM, Ri. = 4300 sites/cell). Computer analysis of the insulin binding data obtained from frog erythrocytes preincubated with insulin or perturbed by concanavalin A was consistent with an effect on Site I only. Insulin-induced receptor regulation promoted a significant 50% reduction in the number of high-affinity sites, and concanavalin A pretreatment resulted in a 2-fold decrease in the affinity of Site I. No significant effect on either the affinity or number of Site II was detected after pretreatment of the frog erythrocytes with insulin or concanavalin A. Under all experimental conditions the two-site model provides a rational basis for explaining the binding data. The excellent ability of the two-site model to fit the experimental binding data and the sensitivity of only the high-affinity sites to physiological or biochemical perturbations suggest that there may be two independent classes of insulin binding sites in the frog erythrocyte.

Molecular characterization of the β-adrenergic receptor of frog erythrocytes

Abstract: The beta-adrenergic receptor of the frog erythrocyte has been solubilized in an active form with digitonin and purified by affinity chromatography and high performance liquid chromatography. Purified preparations contain a single band of iodinated protein of apparent Mr = 58,000. This peptide appears to represent the ligand binding subunit of the receptor since purified preparations bind ligands with the same beta-adrenergic specificity as the solubilized or membrane-bound receptor, display the same isoelectric point and similar sedimentation characteristics in sucrose density gradients. The same ligand binding subunit can also be identified in partially purified receptor preparations or in membranes by photoaffinity labelling or photodependent crosslinking of two radiolabelled beta-adrenergic antagonists, p-azidobenzylcarazolol and p-aminobenzylcarazolol.