TY - JOUR
T1 - Fluorescence resonance energy transfer analysis of secretin docking to its receptor
T2 - Mapping distances between residues distributed throughout the ligand pharmacophore and distinct receptor residues
AU - Harikumar, Kaleeckal G.
AU - Lam, Polo C.H.
AU - Dong, Maoqing
AU - Sexton, Patrick M.
AU - Abagyan, Ruben
AU - Miller, Laurence J.
PY - 2007/11/9
Y1 - 2007/11/9
N2 - Full structural characterization of G protein-coupled receptors has been limited to rhodopsin, with its uniquely stable structure and ability to be crystallized. For other members of this important superfamily, direct structural insights have been limited to NMR structures of soluble domains. Two members of the Class II family have recently had the structures of their isolated amino-terminal regions solved by NMR, yet it remains unclear how that domain is aligned with the heptahelical transmembrane bundle domain of those receptors. Indeed, three distinct orientations have been suggested for different members of this family. In the current work, we have utilized fluorescence resonance energy transfer to establish the distances between four residues distributed throughout fully biologically active, high affinity analogues of secretin and distinct residues in each of four extracellular regions of the intact secretin receptor. These 16 distance constraints were utilized along with nine photoaffinity labeling spatial approximation constraints to study the three proposed orientations of the peptide-binding amino terminus and helical bundle domains of this receptor. In the best model, the carboxyl terminus of secretin was found to bind in a groove above the β-hairpin region of the receptor amino terminus, with its amino-terminal end adjacent to the third extracellular loop and top of transmembrane segment VI. This refined model of the intact receptor was also fully consistent with the spatial approximation of the Trp48-Asp49-Asn50 endogenous agonist segment with the third extracellular loop region that it has been shown to photolabel. This provides strong evidence for the orientation of peptide-binding and signaling domains of a prototypic Class II G protein-coupled receptor.
AB - Full structural characterization of G protein-coupled receptors has been limited to rhodopsin, with its uniquely stable structure and ability to be crystallized. For other members of this important superfamily, direct structural insights have been limited to NMR structures of soluble domains. Two members of the Class II family have recently had the structures of their isolated amino-terminal regions solved by NMR, yet it remains unclear how that domain is aligned with the heptahelical transmembrane bundle domain of those receptors. Indeed, three distinct orientations have been suggested for different members of this family. In the current work, we have utilized fluorescence resonance energy transfer to establish the distances between four residues distributed throughout fully biologically active, high affinity analogues of secretin and distinct residues in each of four extracellular regions of the intact secretin receptor. These 16 distance constraints were utilized along with nine photoaffinity labeling spatial approximation constraints to study the three proposed orientations of the peptide-binding amino terminus and helical bundle domains of this receptor. In the best model, the carboxyl terminus of secretin was found to bind in a groove above the β-hairpin region of the receptor amino terminus, with its amino-terminal end adjacent to the third extracellular loop and top of transmembrane segment VI. This refined model of the intact receptor was also fully consistent with the spatial approximation of the Trp48-Asp49-Asn50 endogenous agonist segment with the third extracellular loop region that it has been shown to photolabel. This provides strong evidence for the orientation of peptide-binding and signaling domains of a prototypic Class II G protein-coupled receptor.
UR - http://www.scopus.com/inward/record.url?scp=36349035022&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=36349035022&partnerID=8YFLogxK
U2 - 10.1074/jbc.M704563200
DO - 10.1074/jbc.M704563200
M3 - Article
C2 - 17827151
AN - SCOPUS:36349035022
SN - 0021-9258
VL - 282
SP - 32834
EP - 32843
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 45
ER -