Self-Controlled Regulation of Mu Opioid Receptor Trafficking

Our cells rely extensively on external signals for key cellular and systematic functions. Many of these signals take the form of small molecules that are recognized by receptors on the cell surface. G-protein coupled receptors (GPCRs) are the largest group of surface signaling receptors. GPCRs respond to a diverse array of signals including: light, pain, adrenaline and serotonin. The capability of a certain type of GPCR to receive its signals is dependent on the number of receptors on the cell surface available to receive that extracellular signal. Intracellular trafficking determines the surface receptor number. Since GPCRs relay such vital signals it has become increasingly clear that their intracellular trafficking is tightly regulated and intertwined with the received and transmitted signals by the GPCRs. This thesis focuses on the clinically, relevant mu-opioid receptor (MOR) which is the primary target of exogenous opiate drugs such as morphine and endogenous opiates such as endorphins and endomorphins. Once activated the MOR internalizes through clathrin-mediated endocytosis. Chapter 1 shows that the MOR is not passive in clathrin-mediated endocytosis, but has the ability to alter the duration of its own endocytosis events. Chapter 2 suggests that this change in endocytosis duration has a functional effect via changes in downstream signaling. Chapter 3 details how protein kinase C (PKC) serves as the control point for two regulatory mechanisms for the postendocytic recycling of the MOR; one stemming from the pain receptor neurokinin-1 and the second downstream of the G-protein signaling of the MOR itself. Chapter 4 explores a proteomic approach using stable isotope labeling of amino acids in cell culture (SILAC) to further elucidate the mechanisms governing MOR endocytosis and recycling.