# Structural and dynamic analysis of GRK interaction with G protein-coupled receptors

> **NIH NIH R01** · THOMAS JEFFERSON UNIVERSITY · 2021 · $528,803

## Abstract

Project Summary
 G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs)
and function to turn off G protein signaling and turn on arrestin-mediated signaling. GRKs have a modular
structure with a central catalytic domain inserted into a regulator of G protein signaling homology (RH) domain,
which itself is bracketed by an N-terminal α-helical domain and a C-terminal lipid-binding region. X-ray
crystallography has revealed that the RH and catalytic domains have extensive contacts with each other and
help to maintain the kinase in an inactive open conformation. To gain mechanistic insight into GRK regulation
of GPCRs, we have studied GRK5 interaction with the β2-adrenergic receptor (β2AR). GRK5 has been
implicated in several diseases including heart failure and hypertension and a GRK5-Q41L polymorphism
prevalent in African Americans has an enhanced ability to desensitize the β2AR and protects against the
development of congestive heart failure. We have identified conditions to generate a β2AR-GRK5 complex to
gain initial insight on the interface of these proteins. Our data support a model that involves a receptor-
mediated disruption of an ionic lock between the GRK5 RH and catalytic domains that is essential for receptor
phosphorylation. We propose to utilize GRK5 interaction with the β2AR as a model to further dissect the
mechanisms involved in GRK activation and gain insight into the normal regulation and function of this
important enzyme family.
 Our central hypothesis is that GRK structure and function is mechanistically linked to interaction with
GPCRs. In aim 1, we will test our initial structural model by mutagenesis and further characterize the dynamics
of β2AR-GRK5 interaction using radiolytic footprinting, double electron-electron resonance spectroscopy and
molecular dynamics simulations. In aim 2, we will use X-ray crystallography and single particle cryo-electron
microscopy to provide insight into the β2AR-GRK5 interface as well as the structural changes that the β2AR
and GRK5 undergo upon binding. We will confirm the binding interface of these proteins and the mechanism of
activation using molecular and biochemical approaches. In aim 3, we will focus on the regulation of GRK5 by
calmodulin. These studies are supported by a crystal structure of a calmodulin-GRK5 complex that reveals that
calmodulin binding disrupts the ionic lock between the RH and catalytic domains and promotes closure of the
catalytic domain. We will verify the calmodulin-GRK5 interface by mutagenesis, further dissect how calmodulin
activates GRK5 and define the role of calmodulin on GRK5 localization and function in cells. Overall, these
studies have broad significance for understanding GRKs and GRK-GPCR interaction and should facilitate the
development of strategies to specifically regulate GRK function in the treatment of disease.

## Key facts

- **NIH application ID:** 10147749
- **Project number:** 5R01HL142310-04
- **Recipient organization:** THOMAS JEFFERSON UNIVERSITY
- **Principal Investigator:** Jeffrey L Benovic
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $528,803
- **Award type:** 5
- **Project period:** 2018-04-01 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10147749

## Citation

> US National Institutes of Health, RePORTER application 10147749, Structural and dynamic analysis of GRK interaction with G protein-coupled receptors (5R01HL142310-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10147749. Licensed CC0.

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