# Methods for mapping cell adhesion receptors

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2023 · $332,539

## Abstract

ABSTRACT
 Transmembrane proteins, which constitute 20%-30% of human genes, play essential roles in coupling
cells and in sensing mechanical and biochemical signals from the environment. However, it is extremely
challenging to map transmembrane protein interactions using traditional biochemical methods. The first goal of
this proposal is to develop Binding Assay for Interacting Transmembrane proteins (BAIT), a molecular technology
to discover novel transmembrane protein interactions in cells. BAIT will be performed in two steps. First, we will
screen for transmembrane proteins that are located proximal to a target protein, by dual tagging both the
extracellular and cytoplasmic region of the target with a proximity labeling enzyme. Next, we will directly test
binding interactions between proximal proteins and the target protein using single molecule Atomic Force
Microscopy (AFM) and also visualize co-localization of the target and binding partner using super-resolution
fluorescence microscopy. We anticipate that BAIT will have a game changing impact in discovering novel
transmembrane junctional proteins interactions on the cell surface.
 The second goal of our proposal is to use BAIT, along with other biophysical tools, to resolve the
assembly and organization of desmosomes, an essential intercellular adhesive organelle that mediates the
integrity of tissues like the epidermis and heart. While mutations in desmosomal proteins are common in
hereditary heart diseases and in skin pathologies, the molecular mechanisms by which these proteins assemble
at the plasma membrane are unknown. Since previous studies show that desmosome formation requires E-
cadherin (Ecad), a ubiquitous cell-cell adhesion protein, we developed a prototype BAIT assay using Ecad as
the target and discovered that two obligate desmosomal adhesive proteins, Desmocollin (Dsc) and Desmoglien
(Dsg), bind to Ecad extracellular regions. Using biophysical experiments and cellular structure function studies
we showed that Ecad recruits Dsg to intercellular contacts, and triggers desmosome formation. In Aim 2 of the
proposal, we will characterize binding interfaces and kinetics of Ecad and Dsc/Dsg interactions and determine
their binding conformations using single molecule AFM binding assays, single molecule Fluorescence
Resonance Energy Transfer and computer simulations. We will also introduce mutant Ecad, Dsc and Dsg in
epithelial cells and monitor desmosome assembly and ultrastructure using super-resolution fluorescence
microscopy. These studies will provide key molecular insights into desmosomal integrity in both healthy tissues
and in disease states.

## Key facts

- **NIH application ID:** 10685306
- **Project number:** 5R01GM121885-08
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** Sanjeevi Sivasankar
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $332,539
- **Award type:** 5
- **Project period:** 2017-02-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10685306

## Citation

> US National Institutes of Health, RePORTER application 10685306, Methods for mapping cell adhesion receptors (5R01GM121885-08). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10685306. Licensed CC0.

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