PROJECT SUMMARY/ABSTRACT
Cholecystokinin acts on type 1 receptors (CCK1Rs) present on vagal afferent neurons to regulate
satiety, important in prevention and treatment of obesity. While CCK1R agonists can acutely reduce feeding,
such agents with high potency and long duration of action tend to be associated with side effects and potential
toxicity not tolerated for chronic therapy of healthy people. It is also now clear that a subset of the population is
refractory to effects of CCK agonists, due to impact of membrane cholesterol on receptor conformation and
dysfunctional stimulus-activity coupling, likely negatively affecting previous clinical trials. Our long term
objective is to target this receptor in a safer and more effective way. The underlying hypothesis is that lack
of mechanistic understanding of interplay between disease states (obesity) and receptor function, and under-
appreciation of novel modes of GPCR drug targeting are key barriers to realizing the therapeutic potential of
CCK1R drugs. Efforts will focus on acquiring molecular understanding of how the membrane environment
affects CCK1R structure and function, and utilizing these insights to pursue opportunities for allosteric
modulation to correct negative impact, and for ligand-directed bias to sculpt signaling and regulatory responses
to activators of this therapeutic target. We will utilize a strong, well-established collaboration, reflecting the
highly complementary skills and experience of Drs. Miller and Sexton. Aim 1 provides a coherent assessment
of signaling and regulatory events initiated by stimulation of CCK1R with orthosteric agonists and allosteric
drugs, and impact of membrane lipids on these events. This provides the opportunity to link distinct ligand
pharmacologies to impact effector engagement, regulatory protein recruitment, control of G protein efficacy,
and receptor sequestration and trafficking, as well as how allosteric cooperativity between sites of molecular
interaction can modify these events. This aim also includes quantification of kinetics of agonist ligand binding
and the events of the G protein cycle that are interdependent, yielding aberrant stimulus-activity coupling with
high binding affinity and reduced signaling at CCK1R in elevated cholesterol. Aim 2 explores the physical
basis for this process, focusing on the site of lipid interaction outside the receptor helical bundle, and its impact
on the mode of natural peptide ligand docking at the ectodomain of this receptor. These studies will utilize
focused chimeric CCK1R:CCK2R constructs, as well as a series of site-directed mutants, and photoaffinity
labeling and fluorescence probing. Aim 3 provides a structural framework for understanding aberrant CCK
stimulus-activity coupling at CCK1R, utilizing our recent structural breakthroughs in determination of active-
state structures of agonist-occupied CCK1R in complex with heterotrimeric G proteins using cryo-EM. These
studies provide a unique paradigm for the investigation of potential drug targets that are affected by the
composition of the plasma membrane in which they reside.