Abstract
The amino-terminal domain of class B G protein-coupled receptors contains six conserved cysteine residues involved in structurally and functionally critical disulfide bonds. The mapping of these bonds has been unclear, with one pattern based on biochemical and NMR structural characterizations of refolded, nonglycosylated amino-terminal fragments, and another pattern derived from functional characterizations of intact receptors having paired cysteine mutations. In the present study, we determined the disulfide bonding pattern of the prototypic class B secretin receptor by applying the same paired cysteine mutagenesis approach and confirming the predicted bonding pattern with proteolytic cleavage of intact functional receptor. As expected, systematic mutation to serine of the six conserved cysteine residues within this region of the secretin receptor singly and in pairs resulted in loss of function of most constructs. Notable exceptions were single mutations of the 4th and 6th cysteine residues and paired mutations involving the 1st and 3rd, 2nd and 5th, and 4th and 6th conserved cysteines, with secretin eliciting statistically significant cAMP responses above basal levels of activation for each of these constructs. Immunofluorescence microscopy confirmed similar levels of plasma membrane expression for each of the mutated receptors. Furthermore, cyanogen bromide cleaved a series of wild type and mutant secretin receptors, yielding patterns that agreed with our paired cysteine mutagenesis results. In conclusion, these data suggest the same pattern of disulfide bonding as that predicted previously by NMR and thus support a consistent pattern of amino-terminal disulfide bonds in class B G protein-coupled receptors.
Highlights
G protein-coupled receptors (GPCRs)1 are important pharmaceutical targets for rational drug design, and have justified a great deal of research aimed at determining the structural aspects of their biological functions
Cyanogen bromide cleaved a series of wild type and mutant secretin receptors, yielding patterns that agreed with our paired cysteine mutagenesis results
Similar to HA37-secretin receptor (SecR), each of the mutant receptors shown was localized to the plasma membrane and inserted such that the amino-terminal HA epitope tag was oriented toward the cell exterior
Summary
Refolded receptor fragments Biochemical, E. coli-expressed amino-terminal domain. GLP-1R 1–3, 2–5, 4–6 a CRFR1/2, corticotropin-releasing factor receptors 1 and 2; SecR, secretin receptor; PTHR1, parathyroid hormone receptor 1; GLP-1R, glucagon-like peptide 1 receptor. It was concluded that because the key structural residues of the fold were conserved among family members, class B receptors likely possess structured aminoterminal domains and bind and signal through the same or closely related mechanisms These very important findings solidified the conclusions of four earlier studies that had deduced the same pattern of disulfide bonds from the sequences and masses of proteolytic fragments derived from corticotropinreleasing factor, glucagon-like peptide 1, and parathyroid hormone receptors [15,16,17,18]. Our findings provide both the definitive disulfide bonding pattern for the clinically important SecR and a needed link between determinations of patterns of disulfide bonds for class B GPCRs that were folded in the cell or refolded in vitro
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