Analysis of candidate cocaine and amphetamine regulated transcript (CART) receptor genes: changes in ERK phosphorylation in N2a cells

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2019
Özkılıç, Ayla
CART is widely expressed in both the central and peripheral nervous system. It encodes CART peptides (CART 55-102 and CART 62-102) that are neurotransmitters and hormones and are positively modulated by leptin which is a satiety hormone. These neuropeptides have important roles in controlling feeding behavior, metabolic rate, hyperphagy, obesity and type 2 diabetes, drug reward, bone remodeling, sensory processing, neuroendocrine function, stress, anxiety, cardiovascular function, gastrointestinal development. Despite the importance of CART, its receptor has not been discovered yet. To understand molecular and cellular pathways of CART, identification of the CART receptor has a high priority. Studies related to binding of CART showed that CART 55-102 induced dose and time dependent activation of extracellular signal-regulated kinase (ERK) 1 and 2. Moreover, inhibition of CART 55-102 by genistein and pertussis toxin indicated that the upstream kinases MEK1 and 2 were involved the signaling pathway. Therefore, a role for the Gi/Go coupled G protein coupled receptor (GPCR) in CART 55-102 signaling was considered. Microarray studies were used to analyze over a thousand GPCR signaling related gene products. Among which, 7 candidate genes that may be a CART receptors were determined. In this study, we aimed to express these 7 different candidate CART receptor genes into N2a cells (mouse neuroblastoma cells), stimulate them with CART 62-102 and analyze ERK 1 and 2 phosphorylation (pERK) using Western Blot. We hypothesized that candidate gene/s might increase pERK in presence of a candidate CART receptor. The results show that p-ERK 1/2 signaling has not been detected when the cells were transfected with 7 candidate genes. In conclusion, further investigations are needed to be performed to confirm possibility of these genes candidacy as a potential CART receptor.