Molecular basis of glucagon-like peptide 1 docking to its intact receptor studied with carboxyl-terminal photolabile probes.
The glucagon-like peptide 1 (GLP1) receptor is a member of Family B G protein-coupled receptors and represents an important drug target for type 2 diabetes. Despite recent solution of the structure of the amino-terminal domain of this receptor and that of several close family members, understanding of the molecular basis of natural ligand GLP1 binding to its intact receptor remains limited. The goal of this study was to explore spatial approximations between specific receptor residues within the carboxyl terminus of GLP1 and its receptor as normally docked. Therefore, we developed and characterized two high affinity, full-agonist photolabile GLP1 probes having sites for covalent attachment in positions 24 and 35. Both probes labeled the receptor specifically and saturably. Subsequent peptide mapping using chemical and proteinase cleavages of purified wild-type and mutant GLP1 receptor identified that the Arg(131)-Lys(136) segment at the juxtamembrane region of the receptor amino terminus contained the site of labeling for the position 24 probe, and the specific receptor residue labeled by this probe was identified as Glu(133) by radiochemical sequencing. Similarly, nearby residue Glu(125) within the same region of the receptor amino-terminal domain was identified as the site of labeling by the position 35 probe. These data represent the first direct demonstration of spatial approximation between GLP1 and its intact receptor as docked, providing two important constraints for the modeling of this interaction. This should expand our understanding of the molecular basis of natural agonist ligand binding to the GLP1 receptor and may be relevant to other family members.
Chen
Quan
Q
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259, USA.
Pinon
Delia I
DI
Miller
Laurence J
LJ
Dong
Maoqing
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping / Q000379:methods; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry
D000596:Amino Acids; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D052216:Glucagon-Like Peptide 1