Project Grant R01DA058020

MECHANISMS AND REGULATIONS OF KAPPA OPIOID RECEPTOR SIGNALING - OPIATES HAVE BEEN WIDELY PRESCRIBED FOR PAIN MANAGEMENT IN THE US, WITH A STAGGERING 142 MILLION PRESCRIPTIONS IN 2020 ALONE. WHILE THESE DRUGS EFFECTIVELY RELIEVE PAIN BY INTERACTING WITH THE MU OPIOID RECEPTOR (MOR), THEIR COMPLEX PHARMACOLOGY HAS LED TO CONCERNING SIDE EFFECTS SUCH AS ABUSE POTENTIAL, RESPIRATORY DEPRESSION, AND ADDICTION RISKS. IN FACT, OPIOID OVERDOSE HAS BECOME A LEADING CAUSE OF DEATH IN THE US, WITH MORE THAN 100,000 REPORTED DEATHS IN 2021. CONSEQUENTLY, THERE IS A GROWING NEED TO EXPLORE ALTERNATIVE TREATMENTS FOR PAIN AND RELATED CONDITIONS. ONE HIGHLY PROMISING THERAPEUTIC TARGET IS THE KAPPA OPIOID RECEPTOR (KOR), WHICH NOT ONLY ADDRESSES PAIN BUT ALSO OFFERS POTENTIAL BENEFITS IN TACKLING AFFECTIVE DISORDERS AND ADDICTION. THIS PROPOSAL AIMS TO IDENTIFY THE MECHANISMS AND REGULATORS THAT CONTROL KOR SIGNALING AND POTENTIALLY OVERCOME THE LIMITATIONS AND SIDE EFFECTS ASSOCIATED WITH TRADITIONAL OPIOIDS BY TARGETING NOVEL SIGNAL TRANSDUCERS. THE KOR SIGNALS THROUGH SEVEN GAI SUBTYPES (GI1, GI2, GI3, GOA, GOB, GZ, AND GGUSTDUCIN (GG)) AND TWO B-ARRESTINS (B-ARRESTIN1 AND B-ARRESTIN2). THESE G PROTEINS CONSIST OF GA, GB (GB1-5), AND GG (GG1-13) SUBUNITS, FORMING HETEROTRIMER COMPLEXES. RECENTLY, WE REPORTED THE STRUCTURES OF KOR IN COMPLEX WITH FOUR DIFFERENT G PROTEIN SUBTYPES (GI1, GOA, GZ, AND GG), WHICH SHED LIGHT ON KOR RECEPTOR ACTIVATION AND SIGNALING. EACH G PROTEIN SUBTYPE HAS BEEN FOUND TO PLAY DISTINCT ROLES IN OPIOID-MEDIATED RESPONSES. FUNCTIONAL CHARACTERIZATION HAS ALSO REVEALED SIGNIFICANT DIFFERENCES AMONG THE FOUR G PROTEIN SUBTYPES. MOREOVER, OUR PRELIMINARY STUDIES HAVE IDENTIFIED THAT DIFFERENT COMBINATIONS OF GB AND GG SUBUNITS CAN SIGNIFICANTLY INFLUENCE THE SIGNALING PROFILE OF INDIVIDUAL GA PROTEINS BY AFFECTING THE STABILITY OF THE GA-GB-GG COMPLEX. ADDITIONALLY, GPCR KINASES (GRKS), SUCH AS GRK2, PLAY A CRUCIAL ROLE IN TERMINATING G PROTEIN SIGNALING, PROMOTING RECEPTOR INTERNALIZATION, AND DEGRADATION. INTERESTINGLY, WE HAVE DISCOVERED THAT GRK2 NOT ONLY DIRECTLY INTERACTS WITH KOR AND PHOSPHORYLATES IT BUT ALSO FORMS A STABLE COMPLEX WITH GB1GG2 AND KOR, SUGGESTING A PREVIOUSLY UNKNOWN ROLE OF GRKS. FURTHERMORE, USING PROXIMITY LABELING (APEX) COMBINED WITH MASS SPECTROMETRY (MS), WE HAVE IDENTIFIED LIGAND-SPECIFIC SIGNALING PROFILES ENGAGED BY SPECIFIC G PROTEIN SIGNALING. THESE FINDINGS INDICATE THAT INDIVIDUAL SIGNALING EVENTS MEDIATED BY DIFFERENT TRANSDUCERS MAY SEPARATELY CONTRIBUTE TO THE THERAPEUTIC EFFICACY AND SIDE EFFECTS ASSOCIATED WITH KOR. OUR CENTRAL HYPOTHESIS IS THAT THE LIGAND-SPECIFIC RESPONSES AT KOR ARE DETERMINED BY COMPLEX, NON-TRADITIONAL SIGNALING NETWORKS AT THE CELLULAR LEVEL. TO TEST THIS HYPOTHESIS, WE PROPOSE THE FOLLOWING STUDIES. AIM 1. DEFINE THE ROLE OF NON-TRADITIONAL REGULATORS IN KOR-G PROTEIN SIGNALING. AIM 2. IDENTIFY THE MOLECULAR DETERMINANTS OF GRK SUBTYPE SELECTIVITY IN KOR SIGNALING. AIM 3. PROFILE PROTEIN-PROTEIN INTERACTION NETWORKS IN LIGAND-DEPENDENT CELLULAR SIGNALING RESPONSE OF KOR. THE GOAL OF THESE STUDIES WILL BE A MORE DETAILED UNDERSTANDING OF THE MOLECULAR PHARMACOLOGY OF KOR, THEREFORE PROVIDING THE OPPORTUNITY FOR GAINING NEW INSIGHTS INTO THE CHEMICAL BIOLOGY OF KOR AND ACCESS TO NEW CHEMICAL MODULATORS AS OPIOID ALTERNATIVES.