Role of cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) in regulating the trafficking and signaling of corticotropin-releasing factor receptor 1

Corticotropin releasing factor (CRF) receptor1 (CRFR1) is associated with psychiatric illness and is a proposed target for the treatment of anxiety and depression. Like many G protein-coupled receptors (GPCRs), CRFR1 harbors a PDZ (PSD95/Disc Large/Zona Occludens 1)-binding motif at the end of its carboxyl terminal tail. The interactions of PDZ proteins with GPCRs are crucial for the regulation of their receptor function. In the present study, we characterize the interaction of the cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) with CRFR1. We show using co-immunoprecipitation that the two proteins interact in human embryonic kidney (HEK293) cells in a PDZ motif-dependent manner. We find that the interaction occurs at the Golgi apparatus and that overexpression of CAL retains a proportion of CRFR1 in the intracellular compartment and prevents trafficking to the cell surface. We also demonstrate a significant reduction in the levels of receptor at the plasma membrane upon CAL overexpression, as well as a reduction in internalization. We find that the overexpression of CAL in HEK293 cells resulted in a significant decrease in CRF-stimulated extracellular-regulated protein kinase 1/2 (ERK1/2) phosphorylation, but has no effect on cAMP signaling mediated by the receptor. This effect was dependent on an intact PDZ motif and knockdown of CAL expression using CAL siRNA results in a significant enhancement in ERK1/2 signaling. We show that CAL contributes to the regulation of CRFR1 glycosylation and utilize glycosylation-deficient CRFR1 mutants to further examine the role of glycosylation in the cell surface trafficking of CRFR1. We find that the mutation of Asn residues 90 and 98 results in a reduction in cell surface CRFR1 that is comparable to the effect of CAL overexpression and that these mutants are retained in the Golgi apparatus. Mutation of Asn residues 90 and 98 also results in a decrease in the efficacy for CRF-stimulated cAMP formation mediated by CRFR1. Taken together, our data suggest that CAL can regulate the anterograde trafficking, the internalization as well as the signaling of CRFR1 via modulating the post-translational modifications that the receptor undergoes at the Golgi apparatus.