Functional corticotropin-releasing factor (CRF) receptors in mouse spleen: evidence from adenylate cyclase studies

Radioligand binding studies have previously identified a high affinity, magnesium-dependent, guanine nucleotide-sensitive binding site for corticotropin-releasing factor (CRF) in mouse spleen. In order to determine the functional nature of these CRF binding sites, we examined the effects of CRF on adenylate cyclase activity in mouse spleen homogenates. The stimulation of adenylate cyclase activity was dependent on time, tissue protein concentration, and guanine nucleotides. CRF-stimulated adenylate cyclase activity was evident in the presence of guanosine-5'-triphosphate (GTP) and its precursor guanosine-5'-diphosphate (GDP) but was not detected in the presence of the hydrolysis-resistant GTP analogs, guanyl-5'-imidodiphosphate [Gpp(NH)p] and guanosine-5'-gamma-thiotriphosphate (GTP-gamma-S). The rank order of potency for CRF analogs and fragments in stimulating adenylate cyclase activity was comparable to their affinities for CRF binding sites in mouse spleen homogenates. The putative receptor antagonist, alpha helical ovine CRF(9-41), did not stimulate adenylate cyclase activity but did attenuate the stimulation by various concentrations of rat/human CRF. In summary, these data demonstrate the functional nature of CRF receptors in mouse spleen as evidenced by CRF stimulation of cAMP production and suggest that this peptide may play a physiological role in regulating immune function.