Role of the second and third intracellular loops of metabotropic glutamate receptors in mediating dual signal transduction activation

On the basis of sequence homology and structural similarities, metabotropic glutamate receptors (mGluRs), extracellular Ca2+-sensing receptor, gamma-aminobutyric acid type B receptor, and pheromone receptors are enlisted in a distinct family within the larger G protein-coupled receptor superfamily, When expressed in heterologous systems, group I mGluRs can activate dual signal transduction pathways, phosphoinositides turnover and cAMP production. To investigate the structural basis of these coupling properties, we introduced single amino acid substitutions within the second and third intracellular loops (i2 and i3) of mGluR1 alpha. Wildtype and mutant receptors were expressed in human embryonic kidney 293 cells and analyzed for their capacity to stimulate both signaling cascades. Each domain appeared to be critical for the coupling to phospholipase C and adenylyl cyclase. Within i2, Thr(695), Lys(697), and Ser(702) were found to be selectively involved in the interaction with G class alpha subunit(s), whereas mutation of Pro(698) and the deletion Cys(694)-Thr(695) affected only G(s) coupling. Furthermore, the mutation K690A profoundly altered mGluR1 alpha signaling properties and imparted to the receptor the ability to couple to the inhibitory cAMP pathway. Within i3, we uncovered two residues, Arg(775) and Phe(781), that are crucial for coupling to both pathways, since their substitution leads to receptor inactivation.