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Biomedical subjects

R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 415 records · Page 23Linked to original sources
PubMed1983

Reconstitution of beta-adrenergic receptors in lipid vesicles: affinity chromatography-purified receptors confer catecholamine responsiveness on a heterologous adenylate cyclase system.

The binding function of purified receptors can be assessed with radioligands, but the interaction of receptors with their biochemical effectors has not been amenable to direct study. Toward this end, procedures have been developed for directly demonstrating functionality of purified beta-adrenergic receptor preparations. Digitonin-solubilized beta-adrenergic receptors from frog erythrocytes or rat lung were purified approximately equal to 100- to 5,000-fold by affinity chromatography and inserted into a mixture of frog erythrocyte lipids and dimyristoyl phosphatidylcholine in the presence of octyl glucoside. Reconstitution of beta-adrenergic receptor binding was typically 25-50% and could also be effected with soybean phosphatidylcholine in the presence of octyl glucoside. The reconstituted beta-adrenergic receptors were then fused with Xenopus laevis erythrocytes, which contain prostaglandin E1-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] but few beta-adrenergic receptors and little or no catecholamine-sensitive adenylate cyclase. Fusion of reconstituted receptor with Xenopus laevis erythrocytes establishes a substantial (2- to 10-fold) stimulation of the hybrid adenylate cyclase by the beta-agonist isoproterenol. The extent of stimulation depends on the amount of reconstituted beta-adrenergic receptor added, is blocked by propranolol, and is eliminated by boiling the beta-adrenergic receptor prior to reconstitution. The successful coupling of a purified receptor to a heterologous adenylate cyclase opens the way to the study of receptor structure---function relationships.

Adenylyl Cyclases↗
PubMed1983

Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain.

Antibodies to the beta 2-adrenergic receptor of frog erythrocytes have been raised in rabbits by immunization with purified receptor preparations. Binding of the antibodies to the receptors was demonstrated by immunoprecipitation and by the altered mobility of the antibody-bound receptors on steric-exclusion HPLC columns. As assessed by a radioimmunoassay developed with the antibody, beta 2-adrenergic receptors from several sources showed various degrees of immunological crossreactivity whereas several beta 1-adrenergic receptors did not crossreact. The antibody appeared to not bind at the ligand binding site of the receptor and did not perturb antagonist radioligand binding to the receptor. Nonetheless, the antibodies selectively attenuated catecholamine-stimulated adenylate cyclase. This suggests that the antibodies recognize and bind to domains of the receptor other than the binding site and that may be involved in coupling to other components of the adenylate cyclase system. Immunocytochemical techniques were used with the antibodies to delineate a postsynaptic localization of beta-adrenergic receptors in rat and frog brain. Thus, these anti-beta-adrenergic receptor antibodies provide a useful reagent for probing beta-adrenergic receptor structure, function, and localization.

Adenylyl Cyclases↗
PubMed1983

The beta-adrenergic receptor in heart failure.

Although the role of the autonomic nervous system in the pathophysiology of heart failure has been studied for decades, only within the past few years have adrenergic receptors and their alterations occupied center stage. There is now considerable evidence linking such receptors to many of the phenomena of heart failure; recent work appears to have significant therapeutic implications.

Adrenergic beta-Agonists↗
PubMed1983

Polymorphonuclear leukocyte function in psoriasis: chemotaxis, chemokinesis, beta-adrenergic receptors, and proteolytic enzymes of polymorphonuclear leukocytes in the peripheral blood from psoriatic patients.

Psoriatic patients, particularly those with psoriatic arthritis, have neutrophilic and eosinophilic leukocytosis. Isolated polymorphonuclear leukocytes (PMNLs) from psoriatic patients have normal concentrations of proteolytic enzymes and they have beta-adrenergic receptors of normal density and affinity. PMNLs from psoriatic patients responded normally to the synthetic chemotactic peptide, f-Met-Leu-Phe (formyl-methionine-leucine-phenylalanine). The chemotactic activities of sera from psoriatic patients were similar to those of normal sera. Sera from psoriatic patients enhanced chemokinesis of PMNLs more than normal control sera at a final concentration of 1%; no difference in chemokinetic response between psoriatic and normal sera was found at serum concentrations greater than 2.5%. This study suggests that the peripheral PMNLs from psoriatic patients are normal, but the sera of psoriatic patients has more chemokinetic activity for PMNLs than does normal serum.

Chemotaxis, Leukocyte↗
PubMed1983

In vitro desensitization of beta adrenergic receptors in human neutrophils. Attenuation by corticosteroids.

The receptor alterations involved in catecholamine-induced desensitization of adenylate cyclase in human neutrophils have been investigated as has the ability of hydrocortisone to modify such alterations. Incubation of human neutrophils with isoproterenol for 3 h in vitro resulted in an 86% reduction in the ability of isoproterenol to stimulate cyclic AMP accumulation in the cells. Two types of receptor alterations were documented. There was a 40% reduction in the number of beta adrenergic receptors (42 vs. 25 fmol/mg protein, P < 0.005) present after desensitization as assessed by [(3)H]dihydroalprenolol ([(3)H]DHA) binding. In addition the receptors appeared to be relatively uncoupled from adenylate cyclase. This uncoupling was assessed by examining the ability of the agonist isoproterenol to stabilize a high-affinity form of the receptor, detected by computer modelling of competition curves for [(3)H]DHA binding. Desensitized receptors were characterized by rightward-shifted agonist competition curves. When hydrocortisone was added to the desensitizing incubations (combined treatment) there was a statistically significant attenuation in the desensitization process as assessed by the ability of isoproterenol to increase cyclic AMP levels in the cells. Although combined treatment did not prevent the decline in receptor number, it did attenuate the uncoupling of the receptors. Combined treatment resulted in competition curves intermediate between the control and the rightward-shifted desensitization curves. Prednisolone was similar to hydrocortisone in attenuating isoproterenol-induced uncoupling. Thus, steroids appeared to attenuate agonist-induced desensitization of the beta adrenergic receptor-adenylate cyclase system by dampening the ability of agonists to uncouple receptors without modifying their ability to promote down-regulation of beta adrenergic receptors.

Adenylyl Cyclases↗
PubMed1983

Guanine nucleotides modulate the binding affinity of the oligopeptide chemoattractant receptor on human polymorphonuclear leukocytes.

The oligopeptide chemoattractant receptor on human polymorphonuclear leukocyte (PMN) membranes exists in two affinity states. Since guanine nucleotides regulate the binding affinity and transductional activity of several other types of receptors, we examined the effect of nucleotides on the binding of N-formyl-methionyl peptides to their receptors on human PMN membranes. The addition of guanylylimidodiphosphate (0.1 mM), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMN membrane preparations reduced the fraction of high-affinity receptors detected in equilibrium binding studies from 21.3 +/- 0.13 to 11.8 +/- 0.05% (P less than 0.03), without altering the binding affinities. Since the total number of receptors remained unchanged, the effect of guanylylimidodiphosphate was to convert a portion of the receptors from the high-affinity state to the low-affinity state. At the maximal concentration of guanine nucleotide tested, approximately 50% of the high-affinity sites were converted to low-affinity sites. The findings obtained by equilibrium binding were supported by kinetic studies since the dissociation of the radiolabeled oligopeptide chemoattractant N-formyl-methionyl-leucyl-[3H]phenylalanine from PMN membranes was accelerated in the presence of guanine nucleotide. The effect of guanine nucleotides was reversed upon washing, indicating that affinity conversion is bidirectional. The guanine nucleotide effects were greatest with nonhydrolyzable derivatives of GTP followed by GTP then guanosine diphosphate. Neither guanosine monophosphate nor any adenine nucleotide tested had an effect on receptor binding. These data suggest a role for guanine nucleotides in the regulation of stimulus-receptor coupling of chemoattractant receptors on human PMN.

Chemotaxis, Leukocyte↗
PubMed1982

The beta 1-adrenergic receptor of the turkey erythrocyte. Molecular heterogeneity revealed by purification and photoaffinity labeling.

The beta 1-adrenergic receptor of turkey erythrocytes has been purified by a combination of affinity and high performance steric exclusion chromatography. These procedures provide preparations with specific activities of greater than 15,000 pmol/mg of protein with an overall recovery of approximately 30% of the receptor activity solubilized from membrane preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated purified receptor reveals two bands of labeled protein with apparent Mr = 40,000 +/- 2,000 and 45,000 +/- 3,000 in a 3-4:1 ratio. These same two peptides can also be labeled specifically and in approximately the same ration in both membranes and purified preparations using the photoaffinity probe 125I-labeled p-azidobenzylcarazolol. When the two purified polypeptides are completely separated by high performance liquid chromatography and subjected to detailed ligand binding studies, identical beta 1-adrenergic specificities are found for the two receptor forms. Preliminary characterization of these two proteins by partial protease digestion suggests a large degree of similarity between them, albeit with some significant differences. These results demonstrate that both purification and photoaffinity labeling identify two polypeptides in turkey erythrocyte membranes as containing a beta 1-adrenergic receptor binding site. The functional and structural relationships of these two forms of the receptor remain to be elucidated.

Affinity Labels↗
PubMed1982

Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase. Structural alterations in the beta-adrenergic receptor revealed by photoaffinity labeling.

Preincubation of turkey erythrocytes with isoproterenol results in an impaired ability of beta-adrenergic agonists to stimulate adenylate cyclase in membranes prepared from these cells. The biochemical basis for this agonist-induced desensitization was investigated using the new beta-adrenergic antagonist photoaffinity label [125I]p-azidobenzylcarazolol ([125I]PABC). Exposure of [125I]PABC-labeled turkey erythrocyte membranes to high intensity light leads to specific covalent incorporation of the labeled compound into two polypeptides, Mr approximately equal to 38,000 and 50,000, as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Incorporation of [125I]PABC into these two polypeptides is completely blocked by a beta-adrenergic agonist and antagonist consistent with covalent labeling of the beta-adrenergic receptor. After desensitization of the turkey erythrocyte by preincubation with 10(-5) M isoproterenol, the beta-adrenergic receptor polypeptides specifically labeled by [125I]PABC in membranes prepared from desensitized erythrocytes were of larger apparent molecular weight (Mr approximately equal to 42,000 versus 38,000, and 53,000 versus 50,000) compared to controls. When included during the preincubation of the erythrocytes with isoproterenol, the antagonist propranolol (10(-5) M) inhibited both agonist-promoted desensitization of the adenylate cyclase and the altered mobility of the [125I]PABC-labeled receptor polypeptides. These data indicate that structural alterations in the beta-adrenergic receptor accompany the desensitization process in turkey erythrocytes.

Adenylyl Cyclase Inhibitors↗