Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes.

Abstract

A fluorescence receptor binding assay, based upon the high-affinity fi-adrenergic receptor antagonist propranolol, is utilized to probe the microenvironment of the antagonist-receptor complex in the frog (Rana catesbeiana) erythrocyte membrane. The technique of steady-state fluorescence depolarization is applied to the propranolol-receptor complex, allowing quantitation of the rotational relaxation time of the complex. It is found that the complex is dynamically constrained at 20'C. However, in the temperature range 6-100C a sharp reversible release of constraint is observed. It is further demonstrated that the addition of drugs that are known to specifically disrupt the cytoskeleton (colchicine, vincristine, and vinblastine) causes a similar but irreversible release of constraint at 200C. Cytochalasin B has a much smaller influence on the rotational mobility of the propranolol-receptor complex than do the other drugs that disrupt the cytoskeleton. Amphotericin B is without effect on the rotational constraint of the complex. Binding of the antagonist 13H~dihydroalprenolol is not influenced by colchicine. A model is proposed which postulates that cytoskeletal elements are linked to the antagonist-receptor complex. Antagonist binding does not result in cytoskeleta release, whereas agonist binding is postulated to lead to dissociation of the agonist-receptor complex from the cytoskeleton, thereby activating adenylate cyclase.The importance of the f3-adrenergic receptor system derives from its ubiquitous distribution. The 13-receptor is functionally involved in regulation of processes as diverse as epithelial chloride transport (1) and central nervous system activity (2).It has long been clear that the fl-adrenergic receptor operates through an associated adenylate cyclase, which activity the receptor modulates (3). The molecular mechanism of the receptor-cyclase linkage has been the subject of considerable speculation, and although a number of hypotheses have been advanced, none have been validated or repudiated. Consequently, it is of considerable interest to employ receptor-specific biophysical probes, which allow clarification of the intimate details of the linkage.Propranolol, 1-(isopropylamino)-3-naphthyloxy-2-propanol, is a well-characterized antagonist for the ,3-adrenergic receptor, to which it binds with high affinity (4-6). Its availability as a tritium-labeled compound has made possible its utilization in the characterization of the receptor.The naphthalene nucleus of propranolol makes it a likely fluorescent substrate for the ,B-adrenergic receptor. Many compounds of this general structural type have been applied in the past as fluorescent probes in biological systems (7), and propranolol itself has been shown to fluoresce. This property has in fact been utilized in various pharmacokinetic studies (8,9). We have previously shown (10, 11) that the propranolol fluorescence is sufficiently intense to allow useful determinationThe publication costs of this article were defrayed in part by page char...

Molecular complexes involved in the regulation of adenylate cyclase.

“…We recently found an association between a subpopulation of this receptor and digitoning shells of rat erythrocytes (unpublished data). Moreover, agonists, but not antagonists, seem to release the receptor from the cytoskeletal constraints, suggesting that this reaction may be required for hormone action (29). Thus, activation of both the N-protein and the f3adrenergic receptor may involve the dissociation of each of these components from its cytoskeletal binding site.…”

Lateral motion of beta receptors in membranes of cultured liver cells.

“…Five-minute preincubation with isoproterenol (16 uM, 37°C) did not affect the mobility or the distribution ofAlp-NBD on the cells (data not shown).-No change was observed for an additional 15 min at 23°C (Table 3). However, after [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] min at 23°C, the fluorescence became more diffuse, and the Rf increased gradually ( Table 3).…”

Propranolol, atenolol, and trifluoperazine reduce the spontaneous occurrence of meiotic diploid products in Saccharomyces cerevisiae.

“…In yeasts, similar diploid meiotic products have resulted due to the interruption of sporulation (9) and as a consequence of cdc5 and cdc14 (8) The data in the literature on the action of these drugs in mammals and in yeasts may be some help in a preliminary interpretation of our data. The ,-blockers (propranolol, in particular, was used) are able to lower the intracellular cAMP level by acting on the regulatory adenylate cyclase subunit (2). Data have also been reported on the ability of propranolol to antagonize calmodulin and the calmodulin-dependent activity of cyclic nucleotide phosphodiesterase (10).…”