Interaction of microtubules and actin with the N-terminus of βPix-bL directs cellular pinocytosis

Abstract

βPix is a Rac/Cdc42 guanine nucleotide exchange factor (GEF) that is known to be a regulator of actin cytoskeleton remodeling. Recently, a novel splicing isoform, βPix-b(L), was identified as an alternative translational product of the βPix-b mRNA with an extended N-terminus comprising a partial calponin homology (CH) domain and a serine-rich (SR) domain. However, the cellular function of βPix-b(L) is largely unknown. In the current study, we analyzed the genomic DNA structure and cellular functions of βPix-b(L). The results of this study demonstrate that βPix is composed of 24 exons and 21 introns spanning around 100kb. RT-PCR experiments revealed that there are two forms of βPix mRNA with distinct 5' UTRs that are the result of alternative splicing of exon 1 and 2 from βPix genomic DNA. In addition, affinity chromatography analysis and a pull-down assay with the N-terminal region of βPix-b(L) revealed that βPix-b(L) interacts with tubulin and actin via its N-terminal CH and SR domains, respectively. Interaction with tubulin enabled βPix-b(L) to bundle the microtubule and form membrane protrusions. Furthermore, the N-terminus of βPix-b(L) was also critical for its localization to cellular vesicles. Functionally, βPix-b(L) induced pinocytosis through cooperative action of the CH and Dbl homology (DH) domains, demonstrating the role of βPix-b(L) in the regulation of membrane dynamics.