Identification and Characterization of a G Protein Coupled Receptor for the Neuropeptide Proctolin. E.C. Johnson ¹, S.F. Garczynski ², D. Nassel ³, D. Park ¹, J.W. Crim ², P.H. Taghert ¹. 1) Dept Anatomy, Neurology, Washington Univ Sch Medicine, St Louis, MO; 2) Dept Cell Biology, University of Georgia, Athens, GA; 3) Department of Zoology, Stockholm University, Stockholm, Sweden.

   We have studied neuropeptide G Protein Coupled Receptors (GPCRs) through functional characterization in mammalian cell lines. Here, we describe evidence that an orphan peptide GPCR is a candidate proctolin receptor. Proctolin is a pentapeptide co-transmitter active on somatic and visceral muscles. After stable integration of the cDNA, HEK-293 cells were loaded with a calcium sensitive fluorescent dye. Baseline fluorescence was measured and a number of synthetic endogenous neuropeptides were added. Only 2 peptides (proctolin and sex peptide) of the 15 tested elicited significant increases in calcium dependant fluorescence at 10-6 M concentrations. Dose responsiveness was assessed and this receptor was sensitive to low concentrations of proctolin (10-10 M) (EC50~ .6 nM) whereas, sex peptide failed to elicit any response in concentrations lower than 10-6 M. Additionally, ¹²⁵I-labeled proctolin was competed away with unlabeled proctolin in isolated membrane preparations derived from the stable cell lines. An antibody was synthesized directed against a synthetic peptide corresponding to the C-terminus and was used to determine receptor expression patterns. By western blot analysis, the antibody detects a ~ 60 kD protein. Proctolin receptor immunoreactivity is consistent with functional data of proctolin sensitivity, and includes the adult hindgut and viscera. Several neuronal populations are also labeled.