Date of Award
Master of Science (MS)
Dr. Laurie B. Cook
Melanin-concentrating hormone (MCH) receptor 1-knockout mice have limited incidence of diet-induced obesity. This makes the MCH signaling pathway a potential pharmacological target to fight human obesity. MCHR1 is a G-protein coupled receptor (GPCR) that activates multiple signaling pathways, including ERK phosphorylation. Overstimulation of GPCR signaling is a hallmark of many diseases. Likewise, inadequate desensitization of MCH signaling could potentiate the obese phenotype. GPCR desensitization typically involves agonist-induced internalization of activated receptors, and subsequent degradation or receptor recycling. The broad aim of this study was to determine the length and intensity of ERK phosphorylation and it's desensitization to MCHR1 activation by MCH. In order to measure this, we maximally stimulated MCHR1-transfected BHK-570 cells with 100 nM MCH for 10 min, then following three washes in serum-free media and a 30 min recovery period, cells were stimulated again. Western blots of lysates for phosphorylated-ERK and total ERK were performed. ImageJ was used to normalize activation levels. MCH was unable to signal a second round of ERK signaling unless we waited 70 minutes, indicating that the MCH signaling pathway is desensitized during this period. We hypothesized that MCHR1 internalization was responsible; however using a cell-based ELISA, we only measured a 15% loss of surface MCHR1 after 30 min of MCH treatment. We tested the hypothesis that G protein-coupled receptor kinases were limiting factors in preventing agonist-mediated endocytosis of MCHR1 however none showed significant gains in internalization. We conclude that MCHRl can undergo receptor-mediated endocytosis, but the fraction of available receptors on the plasma membrane does not account for the extensive loss of ERK signaling observed. We also tested the effect that a GRK2 dominant negative would have on MCHR1 desensitization. In a co-transfected BHK-570 model, we did not observe desensitization if GRK2 is not present. This suggests that GRK2 is necessary for MCHR1 desensitization at the plasma membrane. We have also observed similar ERK desensitization following both isoproterenol treatment and MCHR2 activation which could suggest that simply the ERK pathway desensitizing is being observed which could be independent of the agonist. This study suggests that MCH-mediated ERK signaling desensitizes while MCHR1 is at the plasma membrane, rather than via removal of the receptor from the cell surface. Future experiments will be aimed at determining whether this ERK pathway desensitization is homologous or heterologous in addition to observing downstream pathways of MCHR1 activation other than ERK.
Goodspeed, Andrew E., "Characterization of Melanin-concentrating Hormone Receptor Desensitization" (2013). Biology Master’s Theses. 83.