Point mutations in the first and third intracellular loops of the glucagon-like peptide-1 receptor alter intracellular signaling.
The glucagon-like peptide-1 receptor (GLP-1R) is a member of the glucagon family of seven transmembrane spanning receptors. To investigate how different portions of the GLP-1R may be important in cAMP and intracellular calcium signaling, single amino acid substitutions in the receptor were made by site directed mutagenesis. Receptor binding, cAMP, and intracellular calcium measurements were made in transfected COS-7 cells. The change of amino acid H180R (His to Arg) in the first intracellular loop caused a decrease in the affinity of binding of GLP-1 from 7 nM in the wild type receptor to 150nM and resulted in a 50% decrease in GLP-1 stimulated cAMP production. In response to 10 nM GLP-1, the receptor's ability to stimulate intracellular calcium was altered from a prolonged to a transient response of the same magnitude. Mutation in the 3rd intracellular loop at position R348G (Arg to Gly) decreased receptor affinity from 7 to 83 nM and nearly abolished cAMP production at all concentrations of GLP-1 tested. The GLP-1 stimulated rise in free intracellular calcium was also diminished and this was reversed when cells were treated with forskolin. These results also indicate that GLP-1R signaling via intracellular calcium is dependent on the receptor's ability to also generate cAMP.
Heller
R S
RS
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston 02114, USA.
Kieffer
T J
TJ
Habener
J F
JF
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D005934:Glucagon / Q000378:metabolism / Q000494:pharmacology*; D052216:Glucagon-Like Peptide 1; D000067757:Glucagon-Like Peptide-1 Receptor; D007668:Kidney; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D017354:Point Mutation; D011498:Protein Precursors / Q000378:metabolism / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D051381:Rats; D018027:Receptors, Glucagon / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014162:Transfection
C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D010446:Peptide Fragments; D011498:Protein Precursors; D018027:Receptors, Glucagon; D011994:Recombinant Proteins; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D002118:Calcium