Demonstration of a specific site of covalent labeling of the human motilin receptor using a biologically active photolabile motilin analog.
The motilin receptor belongs to a group of class I G protein-coupled receptors that also includes the growth hormone secretagogue and ghrelin receptors. These represent clinically useful targets for pharmacotherapy. Their potentially unique structures and the molecular basis of their binding are not yet clear. We previously reported the initial affinity labeling of a region within this receptor (a cyanogen bromide fragment extending from the first to the second extracellular loop) using a position 1 photolabile motilin analog. To extend our understanding of the molecular basis of motilin binding, we have developed an additional radioiodinatable motilin analog probe having site of covalent attachment in position 5. This was a full agonist that bound to the motilin receptor specifically and with high affinity, and that efficiently established a single covalent bond to its receptor. Sequential chemical and enzymatic cleavage of labeled wild-type and mutant motilin receptor constructs established that the region of labeling was within the third extracellular loop. This was further localized to Phe(332) using radiochemical Edman degradation sequencing. These data provide the first spatial approximation constraint that can be used in the docking of this peptide ligand to its receptor. We hope that a series of such constraints can be determined to provide adequate structural information to begin to elucidate the conformation of this agonist-bound receptor and to ultimately be useful in the rational design of drugs acting at this important target.
Matsuura
Bunzo
B
Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic in Scottsdale, AZ 85259, USA.
Dong
Maoqing
M
Coulie
Bernard
B
Pinon
Delia I
DI
Miller
Laurence J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D006801:Humans; D008969:Molecular Sequence Data; D009037:Motilin / Q000031:analogs & derivatives* / Q000378:metabolism; D019920:Photoaffinity Labels; D011487:Protein Conformation; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry* / Q000378:metabolism; D018013:Receptors, Neuropeptide / Q000737:chemistry* / Q000378:metabolism
D019920:Photoaffinity Labels; D011964:Receptors, Gastrointestinal Hormone; D018013:Receptors, Neuropeptide; C050802:motilin receptor; D009037:Motilin