A cryptic sequence targets the adhesion complex scaffold ANKS4B to apical microvilli to promote enterocyte brush border assembly

Maura J Graves,Samaneh Matoo,Myoung Soo Choi,Zachary A Storad,Rawnag A El Sheikh Idris,Brooke K Pickles,Prashun Acharya,Paula E Shinder,Taylen O Arvay,Scott W Crawley

doi:10.1074/jbc.ra120.013790

Abstract

Nutrient-transporting enterocytes interact with their luminal environment using a densely packed collection of apical microvilli known as the brush border. Assembly of the brush border is controlled by the intermicrovillar adhesion complex (IMAC), a protocadherin-based complex found at the tips of brush border microvilli that mediates adhesion between neighboring protrusions. ANKS4B is known to be an essential scaffold within the IMAC, although its functional properties have not been thoroughly characterized. We report here that ANKS4B is directed to the brush border using a noncanonical apical targeting sequence that maps to a previously unannotated region of the scaffold. When expressed on its own, this sequence targeted to microvilli in the absence of any direct interaction with the other IMAC components. Sequence analysis revealed a coiled-coil motif and a putative membrane-binding basic-hydrophobic repeat sequence within this targeting region, both of which were required for the scaffold to target and mediate brush border assembly. Size-exclusion chromatography of the isolated targeting sequence coupled with in vitro brush border binding assays suggests that it functions as an oligomer. We further show that the corresponding sequence found in the closest homolog of ANKS4B, the scaffold USH1G that operates in sensory epithelia as part of the Usher complex, lacks the inherent ability to target to microvilli. This study further defines the underlying mechanism of how ANKS4B targets to the apical domain of enterocytes to drive brush border assembly and identifies a point of functional divergence between the ankyrin repeat-based scaffolds found in the IMAC and Usher complex.

Highlights

Intestinal enterocytes undergo an intricate morphogenic program to become specialized to mediate nutrient absorption in the gut
We have previously shown that all the other known intermicrovillar adhesion complex (IMAC) components are largely absent from the early time points of CACO-2BBE cell development [8, 11], with protein levels only beginning to accumulate at the onset of brush border (BB) assembly (Fig. 1A; see CALML4 as example)
Internal deletion of the serinerich patch (SRP) often resulted in the protein becoming restricted to the distal tips of BB microvilli. These results demonstrate that the CC and BHB motifs are necessary for the targeting activity of the ANKS4B Linker segment (LK) segment, whereas the SRP could possibly play a role in regulating incorporation of ANKS4B at the distal tips of BB microvilli

Summary

Introduction

Intestinal enterocytes undergo an intricate morphogenic program to become specialized to mediate nutrient absorption in the gut. We used CACO-2BBE cells to perform structure–function studies and discovered that ANKS4B localizes to apical microvilli using a cryptic targeting sequence found in a previously unannotated region of the protein.

Results

Conclusion