Snapin interacts with G-protein coupled receptor PKR2.

Mutations in Prokineticin receptor 2 (PKR2), a G-protein-coupled receptor, have been identified in patients with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, characterized by delayed puberty and infertility. In this study, we performed yeast two-hybrid screening by using PKR2 C-terminus (amino acids 333-384) as a bait, and identified Snapin as a novel interaction partner for PKR2. The interaction of Snapin and PKR2 was confirmed in GST pull-down and co-immunoprecipitation studies. We further demonstrated that two α-helix domains in Snapin are required for the interaction. And the interactive motifs of PKR2 were mapped to YFK (343-345) and HWR (351-353), which shared a similar sequence of two aromatic amino acids followed by a basic amino acid. Disruption of Snapin-PKR2 interaction did not affect PKR2 signaling, but increased the ligand-induced degradation, implying a role of Snapin in the trafficking of PKR2.

Functional analysis of the distal region of the third intracellular loop of PROKR2.

Mutations in the G-protein-coupled receptor PROKR2 have been identified in patients with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) manifesting with delayed puberty and infertility. Recently, the homozygous mutation V274D was identified in a man displaying KS with an apparent reversal of hypogonadism. The affected amino acid, valine 274, is located at the junction region of the third intracellular loop (IL3) and the sixth transmembrane domain (TM6). In this study, we first studied the effect of V274D and related mutations (V274A, V274T, and V274R) on the signaling activity and cell surface expression of PROKR2. Our data indicate that a charged amino acid substitution at residue 274 of PROKR2 results in low cell surface expression and loss-of-function. Furthermore, we studied the effects of two clusters of basic amino acids located at the proximal region of Val274 on the cell surface expression and function of PROKR2. The deletion of RRK (270-272) resulted in undetectable cell surface expression, whereas RKR (264-266)-deleted PROKR2 was expressed normally on the cell surface but showed loss-of-function due to a deficiency in G-protein coupling. Our data indicate that the distal region of the IL3 of PROKR2 may differentially influence receptor trafficking and G-protein coupling.