G protein-coupled receptors (GPCRs) mediate hormonal control of numerous intracellular effectors. An important mechanism for controlling GPCR signaling involves stimulus-dependent phosphorylation of the receptor, a process primarily mediated by G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of arrestins, which function in receptor/G protein uncoupling and subsequent GPCR endocytosis. GRKs are modular proteins consisting of an N-terminal RGS homology (RH) domain, a central protein kinase catalytic domain, and a C-terminal lipid-binding domain. In this application, we propose to continue our broad-based studies aimed at defining the role of GRKs in regulating cell signaling. Three specific objectives are proposed. 1. Elucidate the molecular mechanism of GPCR binding and activation of GRKs. While GRKs specifically bind to agonist-occupied GPCRs, little is known about the specific residues that mediate receptor binding or how such binding results in GRK activation. We propose two major lines of investigation to test the hypothesis, that GPCR binding is primarily mediated by the catalytic domain. The first will involve generating a series of point mutations based on the recently solved X-ray structure of GRK2. These mutants will be expressed, purified, and analyzed for their ability to bind and phosphorylate various receptor and non-receptor substrates. The second line of investigation will involve expressing and characterizing a catalytic domain construct of GRK2. Taken together these studies should enable us to better define the residues that mediate GRK interaction with GPCRs and elucidate the mechanisms involved in GRK activation. 2. Characterize the functional role of the GRK RH domain. While recent studies have demonstrated that the GRK RH domain interacts with Galphaq family members, the specificity and functional consequences of this interaction remain poorly defined. Interestingly, preliminary studies reveal that GRK2 and GRK3 specifically interact with Galphaq, alpha11, and alphal4 while GRK4 family members interact selectively with Galpha16. The molecular basis for these differences will be further elucidated by site directed mutagenesis and functional analysis of the various GRK RH domains. In addition, we will test the hypothesis that the GRK RH domain functions as an effector for Gaq family members and will test this by characterizing the effects of wild type and Galpha-binding defective GRKs on agonist-dependent phosphorylation and signaling of several GPCRs. 3. Characterize the biology of GRKs using C. elegans as a model organism. C. elegans has served as a powerful model to study a wide variety of biological processes. Interestingly, the C. elegan's genome encodes approximately 1200 GPCRs, 21Galpha subunits, and 12 RGS proteins but only 2 GRKs and a single arrestin. In an effort to better define the biological role of GRKs and correlate structural features with in vivo function, we propose to study the C. elegans GRKs. GRK biology will be studied using knockdown (RNAi and chemical mutagenesis) and transgenic (overexpression of wild type and mutant GRKs) approaches and subsequent analysis of basic biological processes such as egg laying, locomotion, chemotaxis,and adaptation. These studies should enable us to define the biological role of the individual GRKs in C. elegans and start to elucidate the role of specific protein interactions in GRK function. Overall, these studies should more clearly define the molecular and biological role of GRKs in regulating cell signaling.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Lograsso, Philip
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Thomas Jefferson University
Schools of Medicine
United States
Zip Code
Chaturvedi, Madhu; Schilling, Justin; Beautrait, Alexandre et al. (2018) Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors. Trends Biochem Sci 43:533-546
Komolov, Konstantin E; Benovic, Jeffrey L (2018) G protein-coupled receptor kinases: Past, present and future. Cell Signal 41:17-24
Luo, Jiansong; Busillo, John M; Stumm, Ralf et al. (2017) G Protein-Coupled Receptor Kinase 3 and Protein Kinase C Phosphorylate the Distal C-Terminal Tail of the Chemokine Receptor CXCR4 and Mediate Recruitment of ?-Arrestin. Mol Pharmacol 91:554-566
Wang, Jianjun; Luo, Jiansong; Aryal, Dipendra K et al. (2017) G protein-coupled receptor kinase-2 (GRK-2) regulates serotonin metabolism through the monoamine oxidase AMX-2 in Caenorhabditis elegans. J Biol Chem 292:5943-5956
Komolov, Konstantin E; Du, Yang; Duc, Nguyen Minh et al. (2017) Structural and Functional Analysis of a ?2-Adrenergic Receptor Complex with GRK5. Cell 169:407-421.e16
DeRita, Rachel M; Zerlanko, Brad; Singh, Amrita et al. (2017) c-Src, Insulin-Like Growth Factor I Receptor, G-Protein-Coupled Receptor Kinases and Focal Adhesion Kinase are Enriched Into Prostate Cancer Cell Exosomes. J Cell Biochem 118:66-73
Komolov, Konstantin E; Bhardwaj, Anshul; Benovic, Jeffrey L (2015) Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases. J Biol Chem 290:20629-47
Kang, Dong Soo; Tian, Xufan; Benovic, Jeffrey L (2014) Role of ?-arrestins and arrestin domain-containing proteins in G protein-coupled receptor trafficking. Curr Opin Cell Biol 27:63-71
Carr 3rd, Richard; Du, Yang; Quoyer, Julie et al. (2014) Development and characterization of pepducins as Gs-biased allosteric agonists. J Biol Chem 289:35668-84
Quoyer, Julie; Janz, Jay M; Luo, Jiansong et al. (2013) Pepducin targeting the C-X-C chemokine receptor type 4 acts as a biased agonist favoring activation of the inhibitory G protein. Proc Natl Acad Sci U S A 110:E5088-97

Showing the most recent 10 out of 126 publications