GPR160 is not a receptor of anorexigenic cocaine- and amphetamine-regulated transcript peptide

Abstract

Cocaine- and amphetamine-regulated transcript peptide (CARTp) is an anorexigenic neuropeptide whose receptor is undisclosed. Previously, we reported the specific binding of CART(61-102) to pheochromocytoma PC12 cells, where CART(61-102) affinity and the number of binding sites per cell corresponded to ligand-receptor binding. Recently, Yosten et al. designated orphan GPR160 as the CARTp receptor, because the GPR160 antibody abolished neuropathic pain and anorexigenic effects induced by CART(55-102) and exogenous CART(55-102) coimmunoprecipitated with GPR160 in KATOIII cells. As no direct evidence that CARTp is a ligand for GPR160 has been described, we decided to verify this hypothesis by testing CARTp affinity to the GPR160 receptor. We investigated the GPR160 expression in PC12 cells since it is cell line known to specifically bind CARTp. Moreover, we examined the specific CARTp binding in THP1 cells, with high endogenous GPR160 expression and GPR160-transfected cell lines U2OS and U-251 MG. In PC12 cells, the GPR160 antibody did not compete for specific binding with ¹²⁵I-CART(61-102) or with ¹²⁵I-CART(55-102), and GPR160 mRNA expression and GPR160 immunoreactivity were not detected. Moreover, THP1 cells did not show any ¹²⁵I-CART(61-102) or ¹²⁵I-CART(55-102) specific binding despite GPR160 detection by fluorescent immunocytochemistry (ICC). Finally, no ¹²⁵I-CART(61-102) or ¹²⁵I-CART(55-102) specific binding in the GPR160-transfected cell lines U2OS and U-251 MG, selected due to their negligible endogenous expression of GPR160, was detected, despite the detection of GPR160 by fluorescent ICC. Our binding studies clearly demonstrated that GPR160 cannot be a receptor for CARTp. Further studies are needed to identify true CARTp receptors.

Keywords: Binding assay; CARTp; GPCR; GPR160; Transfection.