Item Details

title

CANNABINOID RECEPTOR INTERACTING PROTEIN 1A (CRIP1A): TRANSLOCATION AND INTERACTION PARTNERS

author

Hughes, Erin Kathleen

abstract

The CB1 cannabinoid receptor (CB1R) is a G protein coupled receptor (GPCR) found in the central nervous system (CNS) that modulates neuroprogenitor development, neural commitment, and neurotransmitter release. Cannabinoid receptor interacting protein 1a (CRIP1a) is an abundant ~18 kDa protein in the CNS, which was recently determined to be a β-barrel protein similar in structure to homologous proteins that serve as intracellular carriers of lipidated proteins. In vitro binding studies demonstrated that recombinant CRIP1a interacts directly with myristoylated Gαi-derived peptides. CRIP1a interacts with the CB1R to modulate its cellular signaling; however, the mechanism by which CRIP1a influences CB1R-Gi heterotrimer interactions in neurons is still unknown. I hypothesized that CRIP1a modulates CB1R signaling via an association with the Gαi subunit following agonist-stimulated release from the GPCR. I tested this hypothesis by treating N18TG2 mouse neuroblastoma cells or detergent-free cell homogenates with CB1R agonist CP55940 (100 nM) and monitoring CRIP1a by immunocytochemistry and western immunoblotting. CRIP1a accumulated rapidly and transiently (30-90 sec) from cytosol to plasma membrane fractions after agonist but not inverse agonist SR141716 (100 nM) treatment. Treating intact cells with agonist and determining protein association by proximity ligation immunoassays indicated that CRIP1a and Gαi3 associate transiently within 0.5 min at or near the plasma membrane. No change occurred in response to SR141716 (100 nM) over a similar time course. These findings suggest that agonist stimulation dissociates Gαi3 from the heterotrimer before it associates with CRIP1a. The CRIP1a-Gαi3 complex increased in the nucleus with the same time course. Collectively, these data demonstrate that CRIP1a is capable of associating with myristoylated-Gαi in neuronal cells in response to an agonist stimulation. This sequestration may have a relevant function beyond the plasma membrane, thereby modifying the cannabinoid signaling in cells that express CRIP1a in addition to CB1R. Understanding the molecular mechanisms of CB1R signaling is vital for leveraging the endocannabinoid system and cannabinoids to treat diseases such as cancer, schizophrenia, and chronic pain. Overall, the work and ideas discussed in this dissertation sought to further the understanding of CRIP1a cell signaling and the potential impacts on CB1R signal modulation.

subject

Cannabinoid receptor

Cell signaling

CRIP1a

G protein

Protein-protein interactions

contributor

Howlett, Allyn C (advisor)

Chen, Rong (committee member)

Hollis, Thomas (committee member)

Furdui, Cristina M (committee member)

date

2025-06-24T08:36:28Z (accessioned)

2025 (issued)

degree

Biochemistry and Molecular Biology (discipline)

embargo

2026-06-23 (terms)

2026-06-23 (liftdate)

identifier

http://hdl.handle.net/10339/111007 (uri)

language

en (iso)

publisher

Wake Forest University

type

Dissertation