# Cannabinoid Receptors and Associated Proteins-Renewal

> **NIH NIH R01** · WAKE FOREST UNIVERSITY HEALTH SCIENCES · 2024 · $572,449

## Abstract

Project Summary
The CB1 cannabinoid receptor (CB1R) regulates neuronal processes critical for retrograde signaling to reduce
excessive neurotransmitter release, neuronal development in fetal and adolescent brain, and neuroprotection.
Cannabinoid Receptor Interacting Protein 1a (CRIP1a) is associated with neurodevelopment, sensory function,
seizures, and mental health (Oliver et al., 2020). CRIP1a is a 10-stranded β-barrel protein in the family of
lipidated-protein carriers (with UNC119 and PDE6δ) (Booth et al., 2021). Our studies demonstrate that CRIP1a
binds myristoylated myrGiα N-terminal 9-mer peptide and holo myrGiα. Based upon our major advance in
knowledge of the structure and function of CRIP1a, we hypothesize that CRIP1a functions to sequester or
shuttle Giα proteins, and that the CRIP1a-Giα interaction can be regulated by the CB1R C-terminus during
the agonist-stimulated G protein cycle, palmitoylation-depalmitoylation, Arl protein regulatory cycles,
and phosphorylation-dephosphorylation to control loading and release of Giα cargo. We propose to
investigate the CB1R associated proteins in the N18TG2 and SH-SY5Y human neuroblastoma cell models that
endogenously express the CB1R, CRIP1a and G proteins, as well as using in vitro experiments using purified
recombinant CRIP1a, myrGiα, Gβγ and CB1R C-terminal, as well as peptides derived therefrom.
The Aims are to determine:
1) selectivity for myrGiα as CRIP1a cargo using fluorescence polarization to monitor the binding of peptides
from lipidated Gz, Gs, Gq, and G12/13, various lipids (particularly myristate, palmitate), Gi peptide length and
amino acid sequence, and to define the mechanism by pulldown assays and X-ray structure determination of
these interactions using purified recombinant CRIP1a and peptides or full-length recombinant myrGiα, Gβγ and
CB1R C-terminal;
2) the interface of CRIP1a with the CB1R-G protein activation cycle (mechanism and cellular localization)
by quantitating CRIP1a-Giα co-immunoprecipitation from neuronal cell homogenates treated with GTPγS, full
(2-AG ether, CP55940, WIN55212-2) and partial (methanandamide, Δ9-THC) agonists, competitive antagonist-
inverse agonist (SR141716), and after Gi ADP-ribosylation with pertussis toxin. Cellular location of the CRIP1a-
Giα interaction will be quantitated using proximity ligation assays and subcellular fractionation;
3) the mechanism(s) for loading and unloading myrGiα into CRIP1a by quantitating the gel shifted CRIP1a-
Giα by co-immunoprecipitation in intact neuronal cells or homogenates, and determining myrGiα-CRIP1a
interaction using recombinant proteins to assess: a) roles of palmitoylation and depalmitoylation, b) regulatory
properties of Arl proteins, and c) regulation by phosphorylation of predicted sites on the surface of CRIP1a.
The results of our investigation of CRIP1a and CB1R at the structural and functional level will inform future
investigation of CRIP1a in models of human health and disease.

## Key facts

- **NIH application ID:** 10843855
- **Project number:** 5R01DA042157-07
- **Recipient organization:** WAKE FOREST UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** ALLYN C HOWLETT
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $572,449
- **Award type:** 5
- **Project period:** 2017-08-15 → 2028-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10843855

## Citation

> US National Institutes of Health, RePORTER application 10843855, Cannabinoid Receptors and Associated Proteins-Renewal (5R01DA042157-07). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10843855. Licensed CC0.

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