EHD1—An EH-Domain-Containing Protein with a Specific Expression Pattern Liat Mintz,* Emilia Galperin,* Metsada Pasmanik-Chor,* Sandra Tulzinsky,* Yael Bromberg,† Christine A. Kozak,‡ Alexandra Joyner,§ AmosFein, ¶ and Mia Horowitz* ,1 * Department of Cell Research and Immunology, †Department of Physiology, Sackler Medical School, and ¶ Department of Embryology, Sackler Medical School, Tel-Aviv University, Ramat-Aviv 69978, Israel; ‡Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 329, Bethesda, Maryland 20892; and §Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, New York 10016 Received December 11, 1998; accepted February 22, 1999 A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed dif- ferentially in testis was isolated from mouse and hu- man. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, includ- ing an EF, a Ca 2 binding motif, and a central coiled- coil structure, as well as a nucleotide binding consen- sus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was desig- nated EHD1 (EH domain-containing 1). In cells in tis- sue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, en- docytic vesicles. Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes, and in specific retinal layers. Results of in situ hybridiza- tion to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches and at day 10.5 in sclerotomes, at various ele- ments of the branchial apparatus (mandible and hy- oid), and in the occipital region. At day 15.5 EHD1 expression peaked in cartilage, preceding hypertro- phy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly con- served between nematode, Drosophila, mouse, and hu- man. Its predicted protein structure and cellular lo- calization point to the possibility that EHD1 participates in ligand-induced endocytosis. © 1999 Academic Press INTRODUCTION The diverse effects growth factors have on cell pro- liferation, differentiation, and metabolism are medi- ated by interaction with cell surface receptors. There are several receptor families that convey their ligand- induced signals through different intracellular mecha- nisms. One family of receptors possesses tyrosine ki- nase activity (Darnell et al., 1995) and includes the EGFR, 2 the insulin receptor, and the IGF1 receptor. Binding of these receptors to their ligands induces a cascade of events, leading to sequestration of the li- gand-bound receptor in endocytic vesicles (Kirch- hausen et al., 1997; Mukherjee et al., 1997; Warren et al., 1998). This process depends on the specific inter- action of clathrin and the clathrin adaptor protein com- plex, AP-2, with specific accessory factors. It has been shown that the EGFR phosphorylates at least two pro- teins, eps15 and eps15R, after which each one of them interacts, through accessory proteins, with AP-2 (Ben- merah et al., 1998; Coda et al., 1998). This interaction leads to endocytosis of the ligand-bound EGFR. Pan1p, the yeast homologue of eps15, was also shown to func- tion as a multivalent adaptor that coordinates protein– protein interactions essential for endocytosis (Wend- land and Emr, 1998). Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. AF099011 (human EHD1 cDNA) and AF099186 (mouse Ehd1 cDNA). 1 To whom correspondence should be addressed at the Department of Cell Research and Immunology, Tel-Aviv University, Ramat-Aviv, Israel. Telephone: 972-3-640 9285. Fax: 972-3-642 2046. E-mail: horwitzm@post.tau.ac.il. 2 Abbreviations used: AP, adaptor protein complex; BBS, Bardet– Biedl syndrome; ECL, enhanced chemiluminescence; EGFR, epider- mal growth factor receptor; EH, eps15 homology; eps, epidermal growth factor receptor pathway substrate; EST, expressed sequence tag; GFP, green fluorescent protein; HRP, horseradish peroxidase; IGF1, insulin-like growth factor 1; ocd, osteochondrodystrophy; ORF, open reading frame; PBS, phosphate-buffered saline. Genomics 59, 66 –76 (1999) Article ID geno.1999.5800, available online at http://www.idealibrary.com on 66 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.