Molecular Characterisation of Small Molecule Agonists Effect on the Human Glucagon Like Peptide-1 Receptor Internalisation.
The glucagon-like peptide receptor (GLP-1R), which is a G-protein coupled receptor (GPCR), signals through both Gαs and Gαq coupled pathways and ERK phosphorylation to stimulate insulin secretion. The aim of this study was to determine molecular details of the effect of small molecule agonists, compounds 2 and B, on GLP-1R mediated cAMP production, intracellular Ca2+ accumulation, ERK phosphorylation and its internalisation. In human GLP-1R (hGLP-1R) expressing cells, compounds 2 and B induced cAMP production but caused no intracellular Ca2+ accumulation, ERK phosphorylation or hGLP-1R internalisation. GLP-1 antagonists Ex(9-39) and JANT-4 and the orthosteric binding site mutation (V36A) in hGLP-1R failed to inhibit compounds 2 and B induced cAMP production, confirming that their binding site distinct from the GLP-1 binding site on GLP-1R. However, K334A mutation of hGLP-1R, which affects Gαs coupling, inhibited GLP-1 as well as compounds 2 and B induced cAMP production, indicating that GLP-1, compounds 2 and B binding induce similar conformational changes in the GLP-1R for Gαs coupling. Additionally, compound 2 or B binding to the hGLP-1R had significantly reduced GLP-1 induced intracellular Ca2+ accumulation, ERK phosphorylation and hGLP-1R internalisation. This study illustrates pharmacology of differential activation of GLP-1R by GLP-1 and compounds 2 and B.
Institute of Life Science 1, Medical School, Swansea University, Singleton Park, Swansea, United Kingdom.
Aiysha
A
Thompson
Institute of Life Science 1, Medical School, Swansea University, Singleton Park, Swansea, United Kingdom.
Jeffrey W
JW
Stephens
Institute of Life Science 1, Medical School, Swansea University, Singleton Park, Swansea, United Kingdom.
Stephen C
SC
Bain
Institute of Life Science 1, Medical School, Swansea University, Singleton Park, Swansea, United Kingdom.
Venkateswarlu
0000-0002-8739-1483
V
Kanamarlapudi
D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis*; D004705:Endocytosis / Q000187:drug effects*; D000077270:Exenatide; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D052216:Glucagon-Like Peptide 1 / Q000378:metabolism / Q000494:pharmacology; D000067757:Glucagon-Like Peptide-1 Receptor / Q000037:antagonists & inhibitors / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D010446:Peptide Fragments / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology*; D014688:Venoms / Q000494:pharmacology; D000097789:Glucagon-Like Peptide-1 Receptor Agonists
D000067757:Glucagon-Like Peptide-1 Receptor; D010446:Peptide Fragments; D010455:Peptides; D054852:Small Molecule Libraries; D014688:Venoms; C083773:exendin (9-39); D052216:Glucagon-Like Peptide 1; D000077270:Exenatide; D000242:Cyclic AMP; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium; D000097789:Glucagon-Like Peptide-1 Receptor Agonists