Project Summary G protein coupled receptors (GPCRs) are family of receptors that translate extracellular cues into intracellular responses by activating heterotrimeric G proteins. GPCRs are involved in many aspects of biology and play critical roles as receptors for neuromodulators, neurotransmitters, and hormones. GPCRs are characterized by their seven transmembrane domains. Despite having so much of their structure embedded in the membrane, most of the known GPCR regulators are cytosolic proteins. Thus, an unanswered question remains does the membrane shape GPCR signaling and is the cell able to control the local membrane landscape to modulate GPCR function. The mu-opioid receptor (µOR) is a prototypical class A GPCR with clinical relevance as it is the molecular target of opioids and drives the analgesic effect of these drugs, as well as the undesirable side effects that make the ongoing opioid abuse epidemic so devastating. In a screen for µOR regulators, we identified the cholesterol binding protein patched domain containing-1 (PTCHD1) as a negative regulator of opioid action. PTCHD1 is a critical regulator of opioid action and its knockout in mice results in elevated opioid efficacy without the development of tolerance. Our data suggests that PTCHD1 acts to shape the local membrane environment reducing the accessibility of cholesterol. Here we explore how PTCHD1 and membrane cholesterol regulate the µOR which will lead to a better understanding of how GPCRs are regulated by the membrane. We hypothesize that PTCHD1 acts in a localized manner to reduce cholesterol accessibility preventing membrane cholesterol from acting as a positive allosteric modulator of the µOR and altering the postendocytic trafficking of the receptor. We will test this hypothesis using a combination of cellular signaling and protein trafficking assays in cell lines as well as in primary neurons. This line of research is predicted to provide novel insights into the mechanisms that shape the action of opioids and underlie the development of tolerance, a major obstacle in the use of opioids in pain management that contributes to the opioid abuse crisis. Further, this research is poised to reveal a novel avenue of GPCR regulation through shaping the membrane lipid composition.