Fig. 5: Overview of cysteine-cross-linking by analogous cysteine-substituted agonist and antagonist secretin peptides.

a Cysteine trapping of secretin receptor ECL3 cysteine replacement mutants with ¹²⁵I-labeled Cys⁵- or Cys⁶-containing secretin antagonist analogs. Shown are typical autoradiographs of 10% SDS–PAGE gels used to separate the products of cysteine trapping of the indicated ECL3 SecR cysteine replacement mutants transiently expressed in COS-1 cells for each of the noted cysteine-containing secretin antagonist probes, under nonreducing (top panel) and reducing (bottom panel) conditions. Autoradiographs are representative of a minimum of three independent experiments. Densitometric analysis of data from three similar experiments for each probe is shown (middle panel), with intensities representing the percentages of the signal for the maximal labeling of a residue within ECL3 by that probe. b Illustration of the difference of labeling within the extracellular loops of the cysteine-substituted secretin receptor mutants by cysteine-containing antagonist (left hand panels) and agonist (right hand panels) probes. Blue colored residues are those with most efficient labeling (>50% of the highest efficiency label), with the highest labeled residue denoted with a blue asterisk. Red colored residues denote those that cross-linked with intermediate efficiency (25–50% of the highest efficiency label). c Schematic illustration of the major shifts in residue labeling between equivalent agonist and antagonist probes.