G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins to mediate important physiological responses. Compounds targeting GPCRs represent approximately one-third of FDA-approved drugs. In humans, hundreds of GPCRs converge onto 16 G? subunits that are grouped into 4 families (Gs, Gi/o, Gq/11, G12/13), and individual receptors select between these subtypes. The selectivity of a GPCR for various G protein transducers defines how it influences cellular behavior. Understanding the structures involved in GPCR-G protein selectivity will yield information that can be applied to drug design and understanding cellular signaling. The key focus of this study is determining the relative contributions of the C-terminus (H5.17-H5.26) and the rest of the G? subunit (HN.1-H5.16) to GPCR-G protein coupling selectivity. Prior work suggests that the distal half of helix 5 (H5), the extreme C-terminus of G? subunits, is the key determinant of GPCR-G protein coupling selectivity. Additional specificity-determining sites have since been predicted based on G protein alignment and GPCR-G protein crystal structure data. This project tests the novel hypothesis that G protein residues proximal to G? distal helix 5 are critical determinants of GPCR-G protein selectivity.
Aim 1 will establish the significance of residues proximal to G? distal helix 5(H5.17-H5.26) in GPCR-G protein coupling selectivity via measurement of direct receptor-G protein interactions. A panel of G? subunits that includes a wild-type G protein from each of the 4 families (Gs-long, Gi1, Gq, and G12), and chimeras where the last 10 amino acids (H5.17-H5.26) of each G? is replaced by those of another (e.g. Gsi) will be utilized. Coupling of heterotrimers to 18 GPCRs implicated human pathophysiology will be measured in the presence and absence of both agonists and nucleotides Studies will be conducted in HEK293 Gs/q/12 or Gs/i/q/12(Gless) knockout cells to minimize interference from endogenous G proteins.
Aim 2 will determine the functionality of GPCR-G protein chimera pairs shown to interact directly in an agonist and nucleotide dependent manner Aim 1. This will be accomplished using assays designed to detect activation-dependent heterotrimer rearrangement/dissociation, G?? subunit release, and second messenger regulation. Unexpectedly, preliminary results suggest that GPCR-G protein selectivity determinants are likely to be distributed throughout the G? subunit. These results suggest that for some receptors the regions outside of distal helix 5 appear to the primary determinants of selectivity and that distal H5 influences GPCR-G protein coupling, but is not sufficient to determine selectivity. Given the strong preliminary data gathered, the experienced mentorship team, and the rigorous training plan, this proposal has a high likelihood of success. The completion of this proposal will provide the applicant training in GPCR biology, which will be the basis for a future career developing psychiatric drugs as an independent physician scientist. The proposed study will establish the significance of residues proximal to G? distal helix 5 in GPCR-G proteins coupling selectivity, and elucidate the structural basis of selectivity for this clinically relevant receptor class.

Public Health Relevance

More than one-third of FDA approved drugs target G protein coupled receptors (GPCRs); these receptors activate G proteins that transduce signals to produce beneficial or harmful bodily effects. Understanding how GPCRS choose which G protein to activate, and thus which pathway to activate, can help us develop drugs that specifically target a desired pathway, producing better results with fewer side effects. This project will aid our understanding of GPCRs-G protein selectivity by studying which structural components of the G protein are important for its interaction with a GPCR.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
1F30GM131672-01
Application #
9683391
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Gibbs, Kenneth D
Project Start
2018-12-18
Project End
2021-12-17
Budget Start
2018-12-18
Budget End
2019-12-17
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Augusta University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Augusta
State
GA
Country
United States
Zip Code
30912