Small GTP Binding Proteins in Gastrointestinal Mucosa

Vanderbilt University Medical Center, Nashville TN

Investigators

Linked publications & trials

Abstract

For the past three decades, we have sought to elucidate mechanisms of vesicle trafficking mediated by Rab small GTPase that regulate apical membrane trafficking of key intestinal epithelial transporters that regulate polarized enterocyte function. These investigations led to the recognition of MYO5B and MYO5A as regulators of trafficking through its interaction with multiple Rab proteins. The interaction with Rab10 is based on the presence of a minor MYO5B splice variant coding for the 30 amino acid Exon D. Our determination that MHV M glycoprotein also interacted with MYO5B Exon D, led us to investigate a possible role for MYO5B+D in regulating M protein trafficking. We have successfully completed our major aims to characterize the regulation of M protein trafficking by MYO5B and Rab10, but we have not been able to demonstrate any obligate requirement for this interaction for MHV assembly or egress. We therefore feel that it is necessary to refocus our efforts on our main interests in regulation of gastrointestinal epithelial cells. Our overall goal in Year 5 of the grant is to establish new approaches to modifying Rab11a and Rab8a dependent trafficking pathways that are responsible for discrete aspects of apical enterocyte trafficking. We will therefore pursue two new initiatives this year: First, we will seek to identify specific small molecule inhibitors of the interaction of Rab11a and Rab8a with MYO5B. We will utilize the yeast 2-hybrid assay screening system developed in the first 4 years of the grant to perform high throughput screens of drug libraries. Candidate hits will be tested for the concentration dependence of their action against Rab11a interaction with MYO5B and high potency inhibitors will then be tested for their ability both to inhibit the interaction of endogenous Rab8a or Rab11a with GFP-MYO5B tail. Second, we will seek to identify Rab11a and Rab8a-specific trafficking pathways for apical transporters and enzymes in intestinal epithelial cells. We have created two novel mouse alleles, LSL-Green Lantern-MYO5B tail(YE/QR) and LSL-Scarlet-MYO5B tail(QL/YC) using internal departmental funds. We hypothesize that blockade of Rab11a dependent versus Rab8a dependent trafficking will lead to specific deficits in trafficking and alterations in epithelial physiology. To that end, we will cross each transgenic allele onto the Villin-CreERT2 driver allele for induction of expression in the intestine. We will also assess the sequestration of Rab8a and Rab11a as well as apically directed transporters (including NHE3, SGLT1 and CFTR) with the dominant negative trafficking inhibitors. These studies will allow us for the first time to assess the importance of Rab11a versus Rab8a dependent recycling pathways on the physiology of intestinal enterocytes in vivo.