Interactions between IP3 receptor and Kras-induced actin-interacting protein

Inositol 1,4,5-trisphosphate receptors (IP3R) function as intracellular channels, facilitating the release of Ca2+ from the endoplasmic reticulum (ER) upon IP3 binding. Before responding to IP3 in intact cells, IP3R must undergo a process called 'licensing,' involving the association with Kras-induced actin-interacting protein (KRAP). The immobility of the licensed IP3R fraction is attributed to its interaction with KRAP, which anchors IP3R to actin filaments near plasma membrane (PM)-ER junctions. Given the reported upregulation of KRAP in cells with mutant Kras, I investigated whether non-mutated Kras influences KRAP expression. Knockdown of endogenous Kras using siRNA in HeLa cells slightly altered KRAP expression, suggesting that native Kras is involved in one of other pathways that regulate KRAP expression. Through co-immunoprecipitation (co-IP) analyses, I demonstrated that all three IP3R subtypes interact similarly with native and over-expressed KRAP in HEK cells. Mutation of a conserved motif (204FF) within KRAP (FFAAKRAP) disrupted the KRAP-IP3R interaction. Cytosolic free [Ca2+] measurements in HEK cells expressing individual IP3R subtypes indicated that KRAP licenses all three subtypes to respond to IP3, though IP3R2 was significantly less dependent on KRAP. Direct interaction between KRAP and IP3R was established using co-IP analyses of purified IP3R and bacterially expressed KRAP. This interaction seemed unaffected by the addition of IP3, Ca2+ or cAMP, but experimental limitations did not allow for a conclusive answer as to whether the interaction is regulated by known modulators of IP3R activity. An N-terminal fragment of KRAP (residues 1-265, KRAP265) was capable of directly binding IP3R. The co-IP of KRAP265 with negligible amounts of native KRAP, confirmed that the native KRAP did not account for the ability of the N-terminal fragment of KRAP to associate with IP3R. Investigating whether KRAP forms oligomers in HEK cells presented challenges in interpreting whether the association of the two differentially tagged versions of KRAP was direct or mediated by IP3R or actin. While it was established that the association persisted in cells lacking IP3R, the potential role of actin in the co-IP could not be ruled out. In summary, my findings indicate that KRAP probably binds all three IP3R subtypes directly, facilitating their responsiveness to IP3. The N-terminus of KRAP is required for this interaction, and a conserved motif (204FF) within KRAP is essential. The influence of endogenous Kras on KRAP expression was found to be moderate. Whether KRAP forms oligomers remains unresolved.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Yousef, Abdelhamid

Advisor dc:contributor.advisor

• Ana, Rossi

• Calcium

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/5a2f29e9-a33e-4e45-a8de-092fd44fe119/download
• http://purl.org/NET/rdflicense/allrightsreserved

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.119724

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/386572