Metabotropic glutamate receptor (mGluR) type 7-mediated neurotransmission depends critically on its regulation by associated molecules, such as kinases, phosphatases and structural proteins. The splice variants mGluR7a and mGluR7b are defined by different intracellular C-termini, and simultaneous or exclusive binding of interacting proteins to these domains modulates mGluR7-mediated signalling. However, molecular determinants defining binding regions for associated proteins within mGluR7 C-termini are mostly unknown. In the present study, we have mapped the binding domains of four proteins [filamin A, protein phosphatase (PP) 1C, protein interacting with protein kinase C (PICK) 1 and syntenin] interacting with the mGluR7b variant, and show that the alternatively spliced distal part of the mGluR7b C-terminus was sufficient for the interactions. By individual substitution of all mGluR7b isoform-specific amino acids with alanine and construction of a series of deletion constructs, residues important for the interactions were identified and binding regions could be defined. Interestingly, mGluR7b contains an unusual PP1C-binding motif, located at the N-terminus of the binding domains for PICK1 and syntenin. Consistently, binding of PP1C and PICK1 or PP1C and syntenin to mGluR7b was not competitive. Furthermore, PICK1, but not PP1C, interacted physically with syntenin. Our results represent a molecular description of the binding mechanisms of four mGluR7-associated proteins, and indicate the formation of ternary protein complexes composed of mGluR7b, PP1C, PICK1 and syntenin.
Abbreviations used: β-gal, β-galactosidase; GST, glutathione S-transferase; mGluR, metabotropic glutamate receptor; PKC, protein kinase C; PICK, protein interacting with PKC; PP, protein phosphatase; for brevity the one-letter system for amino acids has been used, e.g. V909 means Val909.