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. Our initial aim was to determine whether MCH signaling desensitizes. 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 when MCH was added to cells, no visible redistribution of MCHR1 was detectable using fluorescence microscopy. We tried a more sensitive assay, a cell-based ELISA, and only measured a 15% loss of surface MCHR1 after 30 min of MCH treatment. Live-cell experiments conducted with rhodamine-MCH and MCHR1-eYFP transfected cells support these conclusions. We tested the hypothesis that β-arrestins and/or GRKs were limiting factors in preventing agonist-mediated endocytosis of MCHR1. Only overexpression of β-arrestins-1 and -2 showed significant gains. We conclude that MCHR1 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. This suggests that MCHR1 mediated ERK signaling desensitizes while MCHR1 is at the plasma membrane, rather than via removal of the receptor from the cell surface. Future experiments are aimed at determining whether this ERK pathway desensitization is homologous or heterologous.
|Presenter:||Andrew Goodspeed (Graduate Student)|
|Time:||11:15 am (Session II)
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