Regulation of Actin Dynamics by Melanin-Concentrating Hormone Potentiates Downstrean Signaling to Extracellular Signal-Regulated Kinase in Cultured Cells

Abstract

Melanin concentrating hormone (MCH) binds and activates two G protein-coupled receptors involved in the control of appetite and energy expenditure in mammals. MCH receptor-I interacts with two cytoskeleton-binding proteins in vitro. One of these proteins, periplakin, has been shown to contribute to the desensitization of MCH signaling events by displacing the interacting G protein. While periplakin is an actin- and intermediate filament-binding protein, MCH is not known to signal cytoskeletal rearrangements. These studies ask whether MCH receptors mediate actin cytoskeletal rearrangements in response to hormone binding. 3T3-L l pre-adipocytes, endogenously expressing MCH receptors, were treated with MCH for varying times, fixed, and actin fibers were stained with Alexa Fluor phalloidin. Using fluorescence microscopy, cells were categorized as 1) having prominent actin stress fibers, 2) being round with many plasma membrane extensions, or 3) being small and round in blinded experiments. Pharmacological agents were used to dissect the contributions of two downstream effectors of Gq, phospholipase C and ADP-ribosylation factor 6, as well as contributions of MCHJ1 versus MCHR2 on MCH-mediated actin rearrangements. A small, but statistically significant change in actin morphology was observed after exposure to MCH for a little as 2 minutes showing MCH docs indeed to the cytoskeleton in this cell line. Phospholipase C activators mimic this response on a similar time course suggesting it is a major participant in this signal transduction pathway.