Harp (harmonin‐interacting, ankyrin repeat‐containing protein), a novel protein that interacts with harmonin in epithelial tissues

Abstract

Mutations in the triple PDZ domain-containing protein harmonin have been identified as the cause of Usher deafness syndrome type 1C. Independently, we identified harmonin in a screen for genes expressed in pancreatic β β β β cells. Using a yeast two-hybrid assay, we show that the first PDZ domain of harmonin interacts with a novel protein, designated harp for h armonin-interacting, a nkyrin r epeat-containing p rotein. This interaction was confirmed in an over-expression system and in mammalian cells, and shown to be mediated by the three C-terminal amino acids of harp. Harp is expressed in many of the same epithelia as harmonin and co-localization of native harp and harmonin was demonstrated by confocal microscopy in pancreatic duct epithelium and in a pancreatic β β β β -cell line. Harp, predicted molecular mass 48 kDa, has a domain structure which includes three ankyrin repeats and a sterile alpha motif. Human harp maps to chromosome 16, and its mouse homologue to chromosome 7. Sequences with similarity to harp include the sans gene, mutations of which are responsible for deafness in the Jackson shaker 2 ( js ) mutant mouse and in human Usher syndrome type 1G. The functional domain structures of harp and harmonin, their interaction under native conditions and their co-localization suggest they constitute a scaffolding complex to facilitate signal transduction in epithelia.

Anks4b, a Novel Target of HNF4α Protein, Interacts with GRP78 Protein and Regulates Endoplasmic Reticulum Stress-induced Apoptosis in Pancreatic β-Cells

“…As shown in Fig. 4E, FLAG-Anks4b was able to coimmunoprecipitate GRP78 as well as harmonin, a protein that was previously found to interact with Anks4b (25). These results indicated that Anks4b binds to GRP78 in cells.…”

“…Reporter gene assays and ChIP analysis demonstrated that HNF4␣ bound to a conserved HNF4 binding motif and activated transcription, thus indicating that Anks4b is a direct target of HNF4␣ in ␤-cells. In addition to the pancreatic islets, Anks4b is also expressed in the liver, kidney, small intestine, and colon (25). This distribution of expression is very similar to that of HNF4␣, suggesting that HNF4␣ plays a role in Anks4b gene transcription in these tissues.…”

“…Anks4b Interacts with GRP78 Both in Vitro and in VivoAnks4b is a scaffold protein with three ankyrin repeats and a sterile ␣ motif domain that was identified as harmonin-interacting protein (25), although its function is completely unknown. To elucidate the role of Anks4b in ␤-cells, we searched for molecules that interacted with full-length Anks4b (FL) and with its deletion mutants (N-, M-, and C-Anks4b) (Fig.…”

Genetics and pathological mechanisms of Usher syndrome

“…11,88,109,114–116 In addition, we and others have shown that the association of other proteins with the USH complex through harmonin indicates that the complex may be functionally linked to a number of basic cell-biological processes, including cell polarity and cell–cell interactions. 117,118 On the basis of the analysis of the whirler mutant and the expression pattern of whirlin in the inner ear, it was hypothesized that whirlin may coordinate F-actin growth. 72 Both CDH23 and PCDH15 are components of transient lateral links, kinociliary links and tip links in the inner ear sensory cells, 89,91,92,119,120 are therefore thought to be crucial for morphogenesis and mechanotransduction.…”

Listen to your gut: Using adhesion to shape the surface of functionally diverse epithelia

“…15,22 ANKS4B was originally identified as a USH1C binding protein that is highly expressed in epithelial tissues. 23 Cell biological studies and biochemical characterization of ANKS4B binding to other IMAC components revealed new insights on the multivalent interactions that drive IMAC assembly, distal tip targeting, and intermicrovillar adhesion. 15 Knockdown of ANKS4B in epithelial cells led to a striking loss of all other IMAC components from microvillar tips; ANKS4B knockdown cells were essentially null for the entire IMAC.…”