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GTPase splice variants RAC1 and RAC1B display isoform-specific differences in localization, prenylation, and

Olivia J Koehn¹, Ellen Lorimer¹, Bethany Unger¹

¹Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.

The Journal of Biological Chemistry

|April 14, 2023

Abstract:

Identifying events that regulate the prenylation and localization of small GTPases will help define new strategies for therapeutic targeting of these proteins in disorders such as cancer, cardiovascular disease, and neurological deficits. Splice variants of the chaperone protein SmgGDS (encoded by RAP1GDS1) are known to regulate prenylation and trafficking of small GTPases. The SmgGDS-607 splice variant regulates prenylation by binding preprenylated small GTPases but the effects of SmgGDS binding to the small GTPase RAC1 versus the splice variant RAC1B are not well defined. Here we report unexpected differences in the prenylation and localization of RAC1 and RAC1B and their binding to SmgGDS. Compared to RAC1, RAC1B more stably associates with SmgGDS-607, is less prenylated, and accumulates more in the nucleus. We show that the small GTPase DIRAS1 inhibits binding of RAC1 and RAC1B to SmgGDS and reduces their prenylation. These results suggest that prenylation of RAC1 and RAC1B is facilitated by binding to SmgGDS-607 but the greater retention of RAC1B by SmgGDS-607 slows RAC1B prenylation. We show that inhibiting RAC1 prenylation by mutating the CAAX motif promotes RAC1 nuclear accumulation, suggesting that differences in prenylation contribute to the different nuclear localization of RAC1 versus RAC1B. Finally, we demonstrate RAC1 and RAC1B that cannot be prenylated bind GTP in cells, indicating that prenylation is not a prerequisite for activation. We report differential expression of RAC1 and RAC1B transcripts in tissues, consistent with these two splice variants having unique functions that might arise in part from their differences in prenylation and localization.

Insights

Small GTPase prenylation and localization are regulated by SmgGDS-607, impacting diseases like cancer. RAC1 and RAC1B splice variants show distinct prenylation and nuclear accumulation, influencing their functions.

Area of Science:

Molecular Biology
Cell Biology
Biochemistry

Background:

Small GTPases are crucial regulators of cellular processes.
Dysregulation of small GTPases is implicated in various diseases, including cancer and cardiovascular disorders.
Splice variants of SmgGDS (RAP1GDS1) modulate small GTPase prenylation and trafficking.

Purpose of the Study:

To investigate the differential effects of SmgGDS-607 binding on the small GTPases RAC1 and its splice variant RAC1B.
To elucidate the mechanisms underlying the distinct prenylation and subcellular localization of RAC1 and RAC1B.
To determine the role of prenylation in the activation of RAC1 and RAC1B.

Main Methods:

Co-immunoprecipitation assays to assess protein-protein interactions.
Western blotting to analyze protein prenylation levels.
Subcellular fractionation and immunofluorescence microscopy to determine protein localization.
Site-directed mutagenesis of the CAAX motif to inhibit prenylation.
GTP binding assays to evaluate protein activation.

Main Results:

RAC1B exhibits more stable association with SmgGDS-607, reduced prenylation, and increased nuclear accumulation compared to RAC1.
The small GTPase DIRAS1 inhibits RAC1 and RAC1B binding to SmgGDS-607 and decreases their prenylation.
Inhibition of RAC1 prenylation promotes its nuclear accumulation, suggesting prenylation differences contribute to localization.
Non-prenylated RAC1 and RAC1B can bind GTP in cells, indicating prenylation is not essential for activation.
Differential expression of RAC1 and RAC1B transcripts suggests unique functional roles.

Conclusions:

SmgGDS-607 binding facilitates RAC1 and RAC1B prenylation, but greater retention of RAC1B by SmgGDS-607 slows its prenylation.
Differences in prenylation and SmgGDS-607 binding contribute to the distinct nuclear localization of RAC1 and RAC1B.
Prenylation is not a prerequisite for RAC1 and RAC1B activation.
The distinct properties of RAC1 and RAC1B splice variants suggest unique physiological functions.

Read full analysis

Keywords:

RAC1B RAP1GDS1 Ras-related C3 botulinum toxin substrate 1 (RAC1)SmgGDS click chemistry nuclear transport protein isoprenylation protein–protein interaction small GTPase

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