Vasopressin-activated Ca²⁺-mobilizing (VACM-1) receptor binds arginine vasopressin (AVP) but does not have amino acid sequence homology with the traditional AVP receptors. VACM-1, however, is homologous with a newly discovered cullin family of proteins that has been implicated in the regulation of cell cycle through the ubiquitin-mediated degradation of cyclin-dependent kinase inhibitors. Because cell cycle processes can be regulated by the transmembrane signal transduction systems, the effects of VACM-1 expression on the Ca²⁺ and cAMP-dependent signaling pathway were examined in a stable cell line expressing VACM-1 in VACM-1 transfected COS-1 cells and in cells cotransfected with VACM-1 and the adenylyl cyclase-linked V₂ AVP receptor cDNAs. Expression of the VACM-1 gene reduced basal as well as forskolin- and AVP-stimulated cAMP production. In cells cotransfected with VACM-1 and the V₂ receptor, the AVP- and forskolin-induced increases in adenylyl cyclase activity and cAMP production were inhibited. The inhibitory effect of VACM-1 on cAMP production could be reversed by pretreating cells with staurosporin, a protein kinase A (PKA) inhibitor, or by mutating S730A, the PKA-dependent phosphorylation site in the VACM-1 sequence. The protein kinase C specific inhibitor Gö-6983 further enhanced the inhibitory effect of VACM-1 on AVP-stimulated cAMP production. Finally, AVP stimulated D-myo-inositol 1,4,5-trisphosphate production both in the transiently transfected COS-1 cells and in the stable cell line expressing VACM-1, but not in the control COS-1 and Chinese hamster ovary cells. Our data demonstrate that VACM-1, the first mammalian cullin protein to be characterized, is involved in the regulation of signaling.