Mechanism of desensitization of adenylate cyclase by lutropin. Impaired introduction of GTP into the regulatory site.

Activity of adenylate cyclase [(EC 4.6.1.1) ATP pyrophosphate lyase (cyclizing)] in purified rat ovarian plasma membranes undergoes partial desensitization (30-60%) upon exposure to saturating lutropin (LH, 0.1 p ~ ) and GTP (1 p ~ ) . Decline in hormone responsiveness of the enzyme is dose-dependent with regard to LH ( K , = 1.7 IWI +. 1) and GTP ( K , = 0.19 p~ f 0.02), indicating that the action of these ligands is probably mediated by the hormone receptor and guanine nucleotide regulatory sites, respectively. The action of GTP in this process is highly specific as demonstrated by the inability of ITP, CTP, or ATP and other nucleotides to substitute for GTP. The desensitizing reaction induced by LH proceeds well in the complete absence of ATP or adenosine 5‘(&y-imido)triphosphate, both substrates for adenylate cyclase. Furthermore, GTP is shown to accelerate desensitization in the presence of adenosine 5’-(a,&methy1ene)triphosphate or CTP, which do not serve as substrates for adenylate cyclase. These findings indicate that continuous catalytic activity of adenylate cyclase is probably not a prerequisite for desensitization. The addition of guanosine 5’-0-(2-thiodiphosphate) (a competitive inhibitor of GTP) results in the cessation of adenylate cyclase activity within seconds. The rate with which this inhibition is achieved is enhanced (3to 4-fold) by LH. In the desensitized state the ability of LH to accelerate this process is reduced along with a similar reduction in the ability of the hormone to stimulate adenylate cyclase. It is suggested that this effect of the hormone provides a new measure for the interaction of the hormone receptor with the guanine nucleotide regulatory site. These results support the view that the function of the hormone is to accelerate the interaction of guanine nucleotides with the regulatory site, thus enhancing the influx of GTP into the GTP-regulatory cycle and, consequently, stimulating adenylate cyclase activity. In the desensitized state this capacity of the hormone is partially lost, resulting in the uncoupling of the enzyme system.