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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.

Clathrin-mediated endocytosis has long been viewed as a process driven by core endocytic proteins, with internalized cargo proteins being passive. In contrast, an emerging view suggests that signaling receptor cargo may actively control its fate by regulating the dynamics of clathrin-coated pits (CCPs) that mediate their internalization. Despite its physiological implications, very little is known about such "cargo-mediated regulation" of CCPs by signaling receptors. Here, using multicolor total internal reflection fluorescence microscopy imaging and quantitative analysis in live cells, we show that the μ-opioid receptor, a physiologically relevant G protein-coupled signaling receptor, delays the dynamics of CCPs in which it is localized. This delay is mediated by the interactions of two critical leucines on the receptor cytoplasmic tail. Unlike the previously known mechanism of cargo-mediated regulation, these residues regulate the lifetimes of dynamin, a key component of CCP scission. These results identify a novel means for selectively controlling the endocytosis of distinct cargo that share common trafficking components and indicate that CCP regulation by signaling receptors can operate via divergent modes.