Characterization of EHD4, an EH domain-containing protein expressed in theextracellular matrix

To identify proteins that promote assembly of type VI collagen tetramers or stabilize type VI collagen filaments, a two-hybrid screen of a human place nta library was used and a new extracellular protein discovered. The cDNA s equence of the new protein encodes 541 amino acid residues. This cDNA seque nce is identical to EHD4, a recently described member of the EH domain fami ly of proteins. Two mRNAs of 4.4 and 3.0 kilobases were present in human sk in fibroblasts and most tissues tested but were most prevalent in the heart . The chromosomal localization of the gene for this new protein was determi ned to be at 15q14-q15. Three polyclonal peptide antibodies were made again st synthetic EHD4 peptides. The affinity-purified antibodies were used in i mmunofluorescent staining of developing limbs and matrices produced by huma n skin fibroblasts and mouse NIH3T3 fibroblasts in culture. Embryonic rat l imb cartilage was strongly stained throughout development, and cultured fib roblasts deposited an extracellular filamentous network containing EHD4. In non-denaturing extracts of fetal bovine cartilage and in human skin fibrob last culture media, two components of similar to 220 and 158 kDa were obser ved, which, after reduction, migrated as a 56-kDa component on SDS-polyacry lamide gel electrophoresis. EHD4 is the first extracellular matrix protein described that contains an EH domain.