# Investigating the molecular details of assembly, disassembly and trafficking of GPCR-arrestin complexes

> **NIH NIH K99** · STANFORD UNIVERSITY · 2023 · $125,000

## Abstract

Project Summary:
Mis-regulation of G protein-coupled receptor (GPCR) trafficking and signaling is implicated in causing several
diseases and the development of drug tolerance, having a major impact on human health. GPCRs evolved to
be the most important means for communication between cells and tissues in higher organisms. They are
responsive to a wide range of stimuli including light, odorants, peptides, neurotransmitters, and hormones,
making GPCRs critical players in regulating human physiology. Owing to their importance, they are the targets
for a third of all FDA-approved drugs. For signaling to be temporally regulated, after agonist stimulation, GPCRs
are desensitized. This desensitization occurs as a two-step process: first by phosphorylation, then by binding to
proteins called -arrestins. -arrestin binding promotes acute desensitization by blocking access of G proteins
to receptors. In addition, -arrestins act as adapters to proteins involved in clathrin-mediated endocytosis,
facilitating internalization of the GPCR. Once internalized, the fate of a GPCR can differ dramatically, from being
rapidly recycled back to the plasma membrane to being degraded. While classically GPCR signaling was thought
to be confined to the plasma membrane, it is now appreciated that GPCRs can also signal from various
intracellular compartments. Though our understanding of G protein-mediated signaling has matured over years
of study, our understanding of how GPCRs are recognized as endocytic cargo remains limited. An important
protein complex for this process is retromer, which sorts cargo at endosomes for recycling. A key component of
retromer, vps26, is structurally similar to -arrestins, and is important for cargo recognition. I hypothesize that
arrestin domain proteins are a privileged scaffold for recognition and trafficking of membrane proteins. As a
result, understanding the molecular mechanisms that determine how GPCR--arrestins assemble and
disassemble, and how they are trafficked in a cell, will have a profound impact on our understanding of signaling
from GPCRs and the action of drugs. Using the 2AR together with V2R and NTSR1 as model receptors, I will
(1) characterize how GPCR--arrestin complexes assemble and disassemble, and how this is affected by
membrane lipids, GPCR phosphorylation, and the presence of other binding partners. I will also (2) identify
protein interaction partners of GPCR--arrestin complexes in cells to understand which factors regulate the rapid
or slow recycling behavior of these receptors. Finally, (3) I will characterize the engagement of a GPCR by
retromer. These aims will be addressed using single-molecule fluorescence spectroscopy, state-of-the-art mass
spectrometry, and in-cell photo-crosslinking. These aims will answer long-standing questions pertaining to
arrestin function, and open new lines of investigation into regulation of GPCRs at endosomes. My Mentor, Dr.
Kobilka, co-mentor Dr. von Zastrow and expert adv...

## Key facts

- **NIH application ID:** 10654850
- **Project number:** 5K99GM147609-02
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** John Janetzko
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $125,000
- **Award type:** 5
- **Project period:** 2022-07-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10654850

## Citation

> US National Institutes of Health, RePORTER application 10654850, Investigating the molecular details of assembly, disassembly and trafficking of GPCR-arrestin complexes (5K99GM147609-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10654850. Licensed CC0.

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