Abstract

Class B1 G protein–coupled receptors are activated by peptides, with amino-terminal regions critical for biologic activity. Although high resolution structures exist, understanding of key features of the peptide activation domain that drive signaling is limited. In the secretin receptor (SecR) structure, interactions are observed between peptide residues His¹ and Ser² and seventh transmembrane segment (TM7) receptor residue E373. We interrogated these interactions using systematic structure-activity analysis of peptide and receptor. His¹ was critical for binding and cAMP responses, but its orientation was not critical, and substitution could independently modify affinity and efficacy. Ser² was also critical, with all substitutions reducing peptide affinity and functional responses proportionally. Mutation of E373 to conserved acidic Asp (E373D), uncharged polar Gln (E373Q), or charge-reversed basic Arg (E373R) did not alter receptor expression, with all exhibiting secretin-dependent cAMP accumulation. All position 373 mutants displayed reduced binding affinities and cAMP potencies for many peptide analogs, although relative effects of position 1 peptides were similar whereas position 2 peptides exhibited substantial differences. The peptide including basic Lys in position 2 was active at SecR having acidic Glu in position 373 and at E373D while exhibiting minimal activity at those receptors in which an acidic residue is absent in this position (E373Q and E373R). In contrast, the peptide including acidic Glu in position 2 was equipotent with secretin at E373R while being much less potent than secretin at wild-type SecR and E373D. These data support functional importance of a charge-charge interaction between the amino-terminal region of secretin and the top of TM7.