Secretin occupies a single protomer of the homodimeric secretin receptor complex: insights from photoaffinity labeling studies using dual sites of covalent attachment.
The secretin receptor, a prototypic family B G protein-coupled receptor, forms a constitutive homodimeric complex that is stable even in the presence of hormone. Recently, a model of this agonist-bound receptor was built based on high resolution structures reported for amino-terminal domains of other family members. Although this model provided the best solution for all extant data, including 10 photoaffinity labeling constraints, a new such constraint now obtained with a position 16 photolabile probe was inconsistent with this model. As the secretin receptor forms constitutive homodimers, we explored whether secretin might dock across both protomers of the complex, an observation that could also contribute to the negative cooperativity observed. To directly explore this, we prepared six secretin analogue probes that simultaneously incorporated two photolabile benzoylphenylalanines as sites of covalent attachment, in positions known to label distinct receptor subdomains. Each bifunctional probe was a full agonist that labeled the receptor specifically and saturably, with electrophoretic migration consistent with labeling a single protomer of the homodimeric secretin receptor. No band representing radiolabeled receptor dimer was observed with any bifunctional probe. The labeled monomeric receptor bands were cleaved with cyanogen bromide to demonstrate that both of the photolabile benzoylphenylalanines within a single probe had established covalent adducts with a single receptor in the complex. These data are consistent with a model of secretin occupying a single secretin receptor protomer within the homodimeric receptor complex. A new molecular model accommodating all constraints is now proposed.
Dong
Maoqing
M
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259.
Lam
Polo C-H
PC
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92037; Molsoft LLC, La Jolla, California 92037.
Pinon
Delia I
DI
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259.
Orry
Andrew
A
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92037; Molsoft LLC, La Jolla, California 92037.
Sexton
Patrick M
PM
Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences, and Department of Pharmacology, Monash University, Parkville 3052, Australia.
Abagyan
Ruben
R
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92037; Molsoft LLC, La Jolla, California 92037.
Miller
Laurence J
LJ
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259. Electronic address: miller@mayo.edu.
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D006728:Hormones / Q000737:chemistry; D008024:Ligands; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics*; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism*; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry* / Q000378:metabolism*; D012633:Secretin / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D006728:Hormones; D008024:Ligands; D010455:Peptides; D021122:Protein Subunits; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; C029937:secretin receptor; D012633:Secretin