Nucleotide regulation of growth hormone-releasing factor binding to rat pituitary receptors

The specific binding of a GRF radioligand, [His1,125I-Tyr10,Nle27]hGRF-1-32NH2, to rat pituitary homogenates is reduced by the addition of GTP and its nonhydrolyzable analogs 5'-guanylylimidodiphosphate (GppNHp) and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). GDP and cAMP had no effect while the nonhydrolyzable ATP analogs 5'-adenylylimidodiphosphate and adenosine 5'-O-(3-thiotriphosphate) did elicit a significant reduction in GRF binding. The effect of GppNHp was half-maximal at 0.2 microM, and the maximum inhibition achieved was 85%. The effect of 0.1 microM GppNHp on GRF competitive displacement experiments indicated a significant reduction in affinity for the ligand (Kd = 0.51 +/- 0.11 nM in the absence of GppNHp and 2.1 +/- 1.1 nM in its presence) without an effect on receptor number. The GRF radioligand dissociates slowly from its receptor (t1/2 = 250 +/- 50 min), but the addition of 0.1 microM GppNHp converts approximately half of the receptors present to a more rapidly dissociating form (t1/2 = 9 +/- 10 min). These results are consistent with existing models for receptor-G-protein interactions, and thus, we conclude that transduction of the GRF response across the cell membrane involves a guanine nucleotide-binding protein, presumably Gs.