CART peptide stimulation of G protein-mediated signaling in differentiated PC12 Cells: Identification of PACAP 6–38 as a CART receptor antagonist

CART peptides are peptide neurotransmitters and hormones that are involved in many different physiological responses. While much is known about the peptides regarding their structure, processing and gene regulation, less is known about their postsynaptic actions and receptors. Using 125I-CART 61–102 as a ligand and unlabeled CART 61–102 or CART 55–102 as displacers, high-affinity specific binding was detected in PC12 cells. Differentiation of the PC12 cells increased specific binding several-fold. The increase in specific binding found after differentiation was inhibited by actinomycin D and cycloheximide, suggesting that the increase in specific binding was dependent on RNA and protein synthesis. CART 1–27, a peptide that has never been shown to elicit responses, did not displace 125I-CART 61–102 binding, nor did more than 20 other peptides that were examined. Surprisingly, however, PACAP 1–38 and PACAP 6–38 were found to be low-affinity inhibitors of CART binding. CART treatment increased binding of 35S-GTPgamma-S to PC12 cell membranes. Moreover, CART treatment of intact PC12 cells elicited robust increases in phospho-ERK in a manner that was increased with differentiation, blocked by pertussis toxin and antagonized by PACAP 6–38. These findings extend previous research and suggest that the CART binding site in PC12 cells reflects a G protein-coupled receptor linked with Gi/o, and also demonstrate that PACAP 6–38 may be useful as a CART receptor antagonist.