Search PubMed⌕ Search

Biomedical subjects

M G Caron

Publications and source records attributed to M G Caron.

At least 379 records · Page 21Linked to original sources
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↗
PubMed1982

Molecular characterization of the beta-adrenergic receptor of frog erythrocytes.

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.

Affinity Labels↗
PubMed1982

Affinity chromatography of human platelet alpha 2-adrenergic receptors.

Catecholamines, such as epinephrine, inhibit the enzyme adenylate cyclase (EC 4.6.1.1) via a specific receptor mechanism involving alpha(2)-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 alpha(2)-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 alpha(2)-adrenergic receptors, can be specifically labeled with [(3)H]yohimbine and can be solubilized with digitonin without loss of their alpha(2)-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 [(3)H]yohimbine binding activity. Adsorption to the affinity gel is blocked by both alpha-adrenergic antagonists (phentolamine >/= yohimbine > prazosin) and by alpha-adrenergic agonists [p-aminoclonidine > (-)-epinephrine > (+)-epinephrine]. Similarly, elution of specific [(3)H]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 alpha-adrenergic receptors, such as (-)-isoproterenol, (-)-alprenolol, atropine, and carbachol, are ineffective for both the blockade of adsorption and the elution of specific [(3)H]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 [(3)H]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 alpha(2)-adrenergic receptor of human platelets.

Adrenergic alpha-Agonists↗
PubMed1982

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

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.

Affinity Labels↗
PubMed1982

Guanine nucleotides regulate both dopaminergic agonist and antagonist binding in porcine anterior pituitary.

Radiolabelled agonist and antagonist binding to porcine anterior pituitary dopaminergic receptor indicates the existence of two affinity forms of the receptor. These two forms of the receptor are modulated by guanine nucleotides. The agonist high affinity form of the receptor is converted to a low affinity form, whereas the antagonist low affinity form is converted to the high affinity form under the influence of guanine nucleotides. Thus, guanine nucleotides appear to reciprocally modulate the same two forms of the receptor discriminated by both agonists and antagonists. These observations may provide insights into the biochemical mechanism by which dopamine exerts its regulation of prolactin release in the anterior pituitary.

Animals↗
PubMed1982

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

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.

Adenylyl Cyclases↗
PubMed1981

Photoaffinity labeling of the beta-adrenergic receptor.

A new photoactive beta-adrenergic antagonist, p-azidobenzylcarazolol (pABC) has been synthesized by combining a carbazole moiety with a p-azido-benzyl substituent. The compound has been labeled with tritium to a specific activity of 26 Ci/mmol. In frog erythrocyte membranes, [3H]p-azido-benzylcarazolol binds to the beta-adrenergic receptor with the expected beta 2 specificity and with high affinity (KD congruent to 100 +/- 10 pM). Unlabeled p-azido-benzylcarazolol can irreversibly inactivate the [3H]dihydroalprenolol-binding activity of frog erythrocyte membranes in a photodependent manner which can be prevented by beta-adrenergic agents. Incubation of frog erythrocyte membranes or digitonin-solubilized preparations of these membranes or digitonin-solubilized preparations of these membranes which had been enriched in beta-adrenergic receptors by a Sepharose-alprenolol chromatography step led to covalent incorporation of radioactivity into a Mr = 58,000 peptide. Specific incorporation of [3H]pABC into the Mr = 58,000 peptide could be prevented by both beta-adrenergic agonists and antagonists. This peptide has previously been purified and shown to contain the beta-adrenergic receptor-binding site (Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826). Thus, photoaffinity labeling of the beta-adrenergic receptor protein directly identifies the same hormone-binding subunit as has been isolated by conventional purification techniques.

Affinity Labels↗
PubMed1980

Macromolecular beta-adrenergic antagonists discriminating between receptor and antibody.

The beta-adrenergic antagonist, alprenolol, was attached in an irreversible manner to macromolecular dextran via side arms that differed in length. The ability of these macromolecules to bind to the beta-adrenergic receptor of frog erythrocytes and to catecholamine-binding antibodies raised against partially purified receptors was studied. Compared to the parent drug the potency of binding of macromolecular alprenolol to the receptor decreased about 1/10, 1/600, and 1/8000 when the length of the arm separating alprenolol from the dextran moiety was 13, 8, and 4 atoms, respectively. In contrast, the binding potencies of the parent drug and of all its macromolecular derivatives for the antibody were within the same order of magnitude. Thus, conversion of a drug to a macromolecular form may not only sustain its binding activity but may also lead in a higher selectivity. The macromolecular derivatives described here may be suitable probes for investigation of the location and of the molecular properties of the binding sites for beta-adrenergic drugs.

Alprenolol↗