# Molecular regulation of the AP2 clathrin adaptor complex

> **NIH NIH R01** · CORNELL UNIVERSITY · 2022 · $249,930

## Abstract

Abstract
Clathrin-mediated endocytosis is the main port of entry into our cells for medically relevant substances including
cholesterol-laden particles and viruses such as influenza and hepatitis. By engulfing signaling receptors, this
fundamental cellular process also tunes our sensitivity to the potentially pathological actions of growth factors
and neuromodulators. As such, understanding how the underlying endocytic machinery is regulated promises to
reveal novel mechanisms that could be harnessed to control neoplastic, neurodegenerative, cardiovascular, and
viral diseases. At the heart of the endocytic process lies the AP2 clathrin adaptor complex which appears to
undergo a conformational change during vesicle formation to actively couple membrane and cargo to the clathrin
coat. Despite the central role of AP2, we lack critical details about how this molecular machine is regulated in
vivo. To address this need, we have developed innovative tools in C. elegans that allow us to quantify AP2
activity at multiple levels and have employed deep genetic screens to identify two conserved protein families that
appear to govern AP2 conformation and activity. Our goal is to illuminate how these allosteric regulators of the
endocytic machinery function mechanistically. Previously it was thought that membrane phospholipids, cytosolic
cargo domains, and phosphorylation by the AP2-associated kinase (AAK1) activate AP2. Our preliminary data
indicate that a conserved region of the membrane-associated Fer/Cip4 Homology Domain-only (FCHo) proteins
is required to promote endocytosis by converting AP2 to an active complex. We have named this functionally
important domain the AP2 Activator, or APA. In Aim 1 we will test whether the APA is sufficient to induce a
structural rearrangement of AP2, as well as defining the roles of membrane, cargo, and phosphorylation in that
process. We will determine where the APA binds to AP2 by screening for C. elegans mutants that escape an
APA anchored to mitochondria. We will evaluate the physiological significance of AP2 phosphorylation by
characterizing kinase mutants. In Aim 2 we will validate our hypothesis that adaptiN-Ear-Binding Coat-
Associated Proteins (NECAP)s counteract the active (open) conformation of AP2 to ensure proper recycling of
adaptor complexes. We have discovered that AP2 accumulates in a hyper-open, hyper-phosphorylated state in
NECAP mutants, and that NECAPs specifically bind open, phosphorylated forms of AP2. We will determine how
NECAPs regulate AP2 activity and where they function within the hierarchy of AP2 modulation using in vitro and
in vivo approaches. To fully understand how NECAPs function, we will determine their structure, and use an
innovative random-scanning mutagenesis technique to determine the relevant NECAP-AP2 contacts in vivo. The
long-term impact of the proposed research will be to clarify how fundamental cellular machinery is controlled with
spatiotemporal precision in metazoans – whe...

## Key facts

- **NIH application ID:** 10582196
- **Project number:** 3R01GM127548-04S1
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** Gunther Hollopeter
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $249,930
- **Award type:** 3
- **Project period:** 2019-04-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10582196

## Citation

> US National Institutes of Health, RePORTER application 10582196, Molecular regulation of the AP2 clathrin adaptor complex (3R01GM127548-04S1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10582196. Licensed CC0.

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