Expression of glucagon receptors in tetracycline-inducible HEK293S GnT1- stable cell lines: an approach toward purification of receptor protein for structural studies.
Glucagon is a 29-amino acid polypeptide hormone secreted by pancreatic A cells. Together with insulin, it is an important regulator of glucose metabolism. Type 2 diabetes is characterized by reduced insulin secretion from pancreatic B cells and increased glucose output by the liver which has been attributed to abnormally elevated levels of glucagon. The glucagon receptor (GR) is a member of family B G protein-coupled receptors, ligands for which are peptides composed of 30-40 amino acids. The impetus for studying how glucagon interacts with its membrane receptor is to gain insight into the mechanism of glucagon action in normal physiology as well as in diabetes mellitus. The principal approach toward this goal is to design and synthesize antagonists of glucagon that will bind with high affinity to the GR but will not activate it. Site-directed mutagenesis of the GR has provided some insight into the interactions between glucagon and GR. The rational design of potent antagonists has been hampered by the lack of structural information on receptor-bound glucagon. To obtain adequate amounts of receptor protein for structural studies, a tetracycline-inducible HEK293S GnT1(-) cell line that stably expresses human GR at high-levels was developed. The recombinant receptor protein was characterized, solubilized, and isolated by one-step affinity chromatography. This report describes a feasible approach for the preparation of human GR and other family B GPCRs in the quantities required for structural studies.
Unson
Cecilia G
CG
Rockefeller University, New York, NY 10065, USA. unsonc@rockefeller.edu
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002087:Butyrates / Q000494:pharmacology; D002460:Cell Line / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004789:Enzyme Activation / Q000187:drug effects; D005240:Feasibility Studies; D005786:Gene Expression Regulation; D005934:Glucagon / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007668:Kidney / Q000166:cytology; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data; D018027:Receptors, Glucagon / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013752:Tetracycline / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D002087:Butyrates; D008024:Ligands; D018027:Receptors, Glucagon; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005934:Glucagon; D000262:Adenylyl Cyclases; D013752:Tetracycline