Biochemical characterization of the cysteine residues within the amino-terminus of the secretin receptor

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The extracellular face of the secretin receptor contains ten cysteine residues, with seven present within the amino-terminal tail, two within the first loop, and one within the second loop domain. In previous mutagenesis studies, we demonstrated that only cysteine residues that are not conserved within the secretin receptor family (Cysll and 186) could be changed to alanines without profound functional impact. It is likely that the conserved cysteine residues are involved in disulfide bonding to establish critical architectural features of the family. In this work, we have used biochemical methods to explore the free versus bonded status of these residues, as well as their functional importance. Secretin ligand binding and stimulated biological activity (cAMP generation) were markedly impaired by treatment of receptor-bearing CHO-SecR cells with cell-impermeant reducing reagents. Insertional mutagenesis was used to introduce an acid-labile Asp-Pro sequence just outside of TM1, to provide a method for the release of the intact, disulfide-bonded amino-terminal tail. This receptor mutant expressed normal binding and biological activity, and was efficiently cleaved with acid treatment. This demonstrated that the amino-terminal domain was not covalently bound to the extracellular loops of this receptor. Stoichiometry of the free versus bonded cysteine residues within this receptor domain was determined by quantitation of [ incorporation in the absence or presence of chemical reduction. This supported the presence of one of seven cysteine residues being free, with the others involved in three intra-domain disulfide bonds. Indeed, treatment of the intact receptor-bearing cells with a cell-impermeant sulfhydrylreactive reagent markedly impaired secretin binding. This effect was markedly reduced when the CysllAla receptor mutant was used in similar studies. Further confirmation of the free nature of this cysteine came from affinity labeling of this region of the receptor using our Bpa probe (J Bioi Chern 1999) and releasing the labeled fragment from the non-reduced receptor with appropriate cleavage. Thus, the amino-terminus of the secretin receptor represents an independent, highly disulfide-bonded domain, with all bonds involving residues that are conserved within this family. The specific nature of these three disulfide bonds should provide useful constraints for the modeling of agonist interaction with this critically important receptor domain.