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Submitted on March 19, 2002
Accepted on July 26, 2002
1 From Center for Basic Research in Digestive Diseases, Departments of Internal Medicine and Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, Rochester, MN 55905 and Vanderbilt University, Department of Chemistry and Center for Structural Biology, Nashville, TN 37232-8725 For consideration of publication in Molec. Endocrinol.
* To whom correspondence should be addressed. E-mail: miller{at}mayo.edu.
The amino terminus of the secretin receptor is known to be critical for natural agonist action, although the role it plays is still unclear. We have demonstrated that photolabile residues within both the amino-terminal (position 6) and carboxyl-terminal (positions 22 and 26) halves of secretin each covalently label receptor amino-terminal tail residues (1
3). Here, we extend this series of studies with an additional probe having its site of covalent attachment in a distinct region of the peptide, between amino- and carboxyl-terminal helical domains. This probe incorporated a photolabile (
-p-benzoylbenzoyl)lysine in position 18 and a site for oxidative radioiodination ([Tyr10,(BzBz)Lys18]rat secretin-27). This analog represented a full agonist, stimulating cAMP accumulation in CHO-SecR cells in a concentration-dependent manner. It bound to the secretin receptor specifically and saturably, and was able to efficiently label that molecule within its amino terminus. Sequential specific cleavage, purification, and sequencing demonstrated that this probe labeled receptor residue Arg14, in the same subdomain as that labeled by previous probes. Consistent with the importance of this residue, Ala replacement mutagenesis (R14A) resulted in substantial reductions in the potency (127-fold) and binding affinity (400-fold) of secretin relative to its action at the wild type receptor. We have been able to accommodate all four extant pairs of residue-residue approximations between divergent regions of the secretin pharmacophore and the first forty residues of the secretin receptor into a credible molecular model of this interaction. Additional experimentally-derived constraints will be necessary to determine the spatial positioning of this complex with the remainder of the secretin receptor.
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