Characterization of EHD4, an EH domain-containing protein expressed in the extracellular matrix.

Huey-Ju Kuo,Noi T Tran,Nicholas P Morris,Robert W Glanville,Susan A Clary

doi:10.1074/jbc.m106128200

Abstract

To identify proteins that promote assembly of type VI collagen tetramers or stabilize type VI collagen filaments, a two-hybrid screen of a human placenta library was used and a new extracellular protein discovered. The cDNA sequence of the new protein encodes 541 amino acid residues. This cDNA sequence is identical to EHD4, a recently described member of the EH domain family of proteins. Two mRNAs of 4.4 and 3.0 kilobases were present in human skin fibroblasts and most tissues tested but were most prevalent in the heart. The chromosomal localization of the gene for this new protein was determined to be at 15q14-q15. Three polyclonal peptide antibodies were made against synthetic EHD4 peptides. The affinity-purified antibodies were used in immunofluorescent staining of developing limbs and matrices produced by human skin fibroblasts and mouse NIH3T3 fibroblasts in culture. Embryonic rat limb cartilage was strongly stained throughout development, and cultured fibroblasts deposited an extracellular filamentous network containing EHD4. In non-denaturing extracts of fetal bovine cartilage and in human skin fibroblast culture media, two components of approximately 220 and 158 kDa were observed, which, after reduction, migrated as a 56-kDa component on SDS-polyacrylamide gel electrophoresis. EHD4 is the first extracellular matrix protein described that contains an EH domain.

Highlights

To identify proteins that promote assembly of type VI collagen tetramers or stabilize type VI collagen filaments, a two-hybrid screen of a human placenta library was used and a new extracellular protein discovered
The affinity-purified antibodies were used in immunofluorescent staining of developing limbs and matrices produced by human skin fibroblasts and mouse NIH3T3 fibroblasts in culture
More than 40 EH domain-containing proteins have been reported [8]. They are intracellular proteins found in organisms ranging from yeast to mammals, most of which have multiple EH domains located in their amino termini, adjacent to a coiled-coil domain

Summary

A New Extracellular Matrix Protein

Mers [11, 12]. Attempts to reconstitute filaments in vitro from purified type VI collagen tetramers failed, so we proposed that other ligands might be required for type VI collagen fibrillogenesis [12]. To find proteins that are potentially involved in type VI collagen filament assembly, the yeast two-hybrid system was used with selected amino- and carboxyl-terminal domains of each ␣(VI) chain to screen a human placenta library [13]. EHD4 was found using a bait peptide composed of most of C1 and C2–C5 domains of the ␣3(VI) carboxyl terminus [14]. We report the complete cDNA sequence of EHD4 and its characterization as an extracellular protein

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION