-
[show abstract]
[hide abstract]
ABSTRACT: This study seeks to investigate the functional state of sequestered beta-adrenoceptors in A431 cells which have undergone homologous desensitization. Incubation of cells with isoprenaline under desensitizing conditions caused a reduction (a) in the number of cell surface beta-receptors as measured by 3H-CGP 12,177 binding, and (b) in the extent of agonist-stimulatable adenylate cyclase activity in membranes prepared from desensitized cells. We infer that those receptors have been sequestered to an internal membrane site since they were detectable by the lipophilic ligand 125I-CYP but not by the more hydrophilic 3H-CGP 12,177. We confirmed that sequestration had occurred by fractionation on non-linear sucrose density gradients of membranes prepared from desensitized cells. The lighter density membrane fraction contained up to 50% of the total receptor pool after desensitization, but only 5-7% of membrane (fluoride-stimulatable) adenylate cyclase. Fusion of desensitized cells in the presence of polyethylene glycol caused a reassociation of sequestered beta-receptors with their biochemical effector Gs since agonist-dependent adenylate cyclase stimulation was restored to the levels measurable after fusion of non-desensitized cells. We conclude that sequestered beta-receptors in desensitized A431 cells are fully functional.
Biochemical Pharmacology 11/1988; 37(19):3701-8. · 4.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Heterologous desensitization of turkey erythrocyte beta-adrenoceptors correlates with receptor phosphorylation and impaired receptor-Gs coupling, as assessed by fusion of purified desensitized receptors with X. laevis erythrocytes [(1984) Science 225, 837-840]. We have purified beta-receptors from desensitized and untreated turkey erythrocytes and have compared the abilities of these two receptors to couple with pure turkey erythrocyte Gs in a reconstituted system. Functional receptor-Gs coupling was assessed by measuring hormone-dependent Gs activation by GTP gamma S and GTPase activity. While in membranes prepared from desensitized cells, receptor-Gs coupling was clearly reduced, this effect was absent when coupling of purified desensitized receptor was measured. We conclude that covalent modification by phosphorylation does not fully explain the functional uncoupling at the membrane level.
FEBS Letters 07/1987; 217(2):287-91. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have previously described a specific protease in turkey erythrocytes that converts the larger 50-kDa (P50) form of the beta 1-adrenoceptor to a smaller 40-kDa (P40) form [Jürss, R., Hekman, M., & Helmreich, E. J. M. (1985) Biochemistry 24, 3349-3354]. Further functional and structural characterization studies of the two forms are reported here. When purified P50 and P40 receptors were compared with respect to their relative capabilities to couple in lipid vesicles with pure stimulatory G-proteins (Gs-proteins) prepared from turkey erythrocytes or rabbit liver, a faster and larger activation of Gs-proteins was observed in response to l-isoproterenol and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with P40 than with P50 receptor. The kon values for P40 were 0.47 min-1 in the case of liver Gs and 0.22 min-1 in the case of erythrocyte Gs, whereas the corresponding values for P50 were 0.34 min-1 and 0.12 min-1, respectively. The binding properties of P50 and P40 forms of the receptor were not different, and desensitization of turkey erythrocytes on exposure to l-isoproterenol did not activate the protease. We furthermore ascertained that only the larger form with a molecular mass of 50 kDa carries the N-linked carbohydrates, which are removed on proteolytic conversion to the 40-kDa form and have either a triantennary or a tetraantennary nonfucosylated complex-type structure containing terminal sialyl residues.
Biochemistry 05/1987; 26(9):2418-25. · 3.42 Impact Factor
