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 mGluR1alpha. Wild-type 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, Thr695, Lys697, and Ser702 were found to be selectively involved in the interaction with Gq class alpha subunit(s), whereas mutation of Pro698 and the deletion Cys694-Thr695 affected only Gs coupling. Furthermore, the mutation K690A profoundly altered mGluR1alpha signaling properties and imparted to the receptor the ability to couple to the inhibitory cAMP pathway. Within i3, we uncovered two residues, Arg775 and Phe781, that are crucial for coupling to both pathways, since their substitution leads to receptor inactivation.
Francesconi
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Margaret M. Dyson Vision Research Institute, Department of Ophthalmology, Cornell University Medical College, New York, New York 10021, USA. afrance@med.cornell.edu
Duvoisin
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D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007545:Isoproterenol / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection / Q000235:genetics; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D018094:Receptors, Metabotropic Glutamate; D010566:Virulence Factors, Bordetella; C104077:metabotropic glutamate receptor type 1; D018698:Glutamic Acid; D000242:Cyclic AMP; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol