A nucleotide regulatory site for somatostatin inhibition of adenylate cyclase in S49 lymphoma cells

Abstract

The cyc− variants of S49 lymphoma cells have served as powerful tools for studying the components and mechanisms of hormone-induced adenylate cyclase stimulation, as these cells are deficient in the guanine nucleotide regulatory site (N_s) mediating hormone, guanine nucleotide, cholera toxin and fluoride-induced stimulations of the enzyme¹. Because of this deficiency, membranes of these cells have been used for reconstitution of the system by inserting the coupling component derived from other cell types^2–4. The hormone-sensitive adenylate cyclase is not only stimulated by hormones but can also be inhibited by a wide variety of hormones and neurotransmitters^5–8, and there is some evidence that hormonal inhibition may be mediated by a distinct guanine nucleotide regulatory site^5–8. Studies in cyc⁻ cells lacking a functional N_s may therefore answer this unresolved, important question. We have recently observed⁹ that stable GTP analogues can inhibit cyc⁻ adenylate cyclase stimulated by purified, preactivated N_s or forskolin, which can activate adenylate cyclase even in the absence of a functional N_s (ref. 10). The data indicated that these N_s-deficient cells contain an inhibitory guanine nucleotide site, N_i. To strengthen this concept, we investigated whether the cyc⁻ adenylate cyclase can be inhibited by a hormone. We report here that somatostatin decreases cyclic AMP levels in cyc⁻ cells, inhibits the forskolin-stimulated adenylate cyclase and causes a concomitant increase in a high affinity GTPase activity in cyc⁻ membranes. The data strongly suggest that both the hormone- and guanine nucleotide-induced adenylate cyclase inhibitions in cyc⁻ cells are mediated by N_i and that the mechanisms of activation and inactivation of N_i are similar to those established for N_s.