Mice with markedly reduced PACAP (PAC(1)) receptor expression by targeted deletion of the signal peptide

In an attempt to study the pituitary adenylate cyclase-activating polypepti de (PACAP) type 1 (PAC(1)) receptor (PAC(1)R) function in vivo and to produ ce a mouse model with altered expression of PAC(1)R, we have used gene targ eting in embryonic stem cells to disrupt exon 2 of the PAC(1)R gene, which contains the ATG translation start site and the signal peptide. Unexpectedl y, active transcription of PAC(1)R mRNA was detected in the mutant mice; ho wever, exon 1 was spliced to exon 3 (skipping exon 2), and I-125-PACAP27 bi nding in brain was greatly reduced. PAC(1)R exon 2(-/-) mice were viable, f ertile, and morphologically and histologically indistinguishable from their wild-type counterparts. We next examined the ligand binding and cell surfa ce expression of the mutant receptor lacking the signal peptide in transfec ted COS-7 cells. I-125-PACAP27 binding of the mutant receptor was approxima tely one-tenth of that in the wild-type receptor. Although the wild-type re ceptor was expressed abundantly in both the plasma membrane and the cytopla sm around the nucleus, the mutant receptor was expressed in the plasma memb rane with a markedly reduced level. Digestion of the membranes with endogly cosidase F greatly reduced the size of the wildtype receptor but only sligh tly reduced that of the mutant receptor. These results demonstrate that the signal peptide is required for efficient cell surface expression and N-lin ked glycosylation of the PAC(1)R. However, the mutant receptors still funct ionally coupled to adenylate cyclase in COS-7 cells, suggesting the presenc e of sufficient spare receptors such that the mutant receptors are capable of activating the second messenger system. We suggest that the mutant mice with markedly reduced PAC(1)R expression can serve as a useful animal model or cell culture system for further studies in PAC(1)R function.