Similarities and Differences in the Way Transmembrane-Type Ligands Interact with the Elk Subclass of Eph Receptors Riccardo Brambilla,* Katja Bru ¨ckner,* Donata Orioli,* Andrew D. Bergemann, †,1 John G. Flanagan, † and Ru ¨ diger Klein* ,2 *European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; and † Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 The Eph family of receptor tyrosine kinases and their cell surface bound ligands have been implicated in a number of developmental processes, including axon pathfinding and fasciculation, as well as patterning in the central nervous system. To better understand the complex signal- ing events taking place, we have undertaken a compara- tive analysis of ligand–receptor interactions between a subset of ligands, those that are tethered to the cell surface via a transmembrane domain, and a subset of Eph receptors, the so-called Elk subclass. Based on binding characteristics, receptor autophosphorylation, and cellu- lar transformation assays, we find that the transmembrane- type ligands Lerk2 and Elf2 have common and specific receptors within the Elk subclass of receptors. The com- mon receptors Cek10 and Elk bind and signal in response to Lerk2 and Elf2, whereas the Myk1 receptor is specific for Elf2. Elf2, however, fails to signal through Cek5 in a cellular transformation assay, suggesting that Lerk2 may be the preferred Cek5 ligand in vivo. A recently identified third transmembrane-type ligand, Elf3, specifically, but weakly, binds Cek10 and only induces focus formation when activated by C-terminal truncation. This suggests that the physiological Elf3 receptor may have yet to be identified. Knowledge regarding functional ligand–recep- tor interactions as presented in this study will be impor- tant for the design and interpretation of in vivo experi- ments, e.g., loss-of-function studies in transgenic mice. INTRODUCTION Signaling by receptor protein-tyrosine kinases (RTKs) controls a wide variety of cellular activities during mammalian development, including proliferation, differ- entiation, cell fate determination, migration, axon target- ing, and cell survival. The largest known subfamily of RTKs is the Eph subfamily, comprising at least 13 distinct members, not including apparent orthologs in different species (for recent reviews see Brambilla and Klein, 1995; Friedman and O’Leary, 1996). Most Eph-related receptors are specifically expressed during embryonic development, many of them in the developing nervous system, suggesting important roles in neurogenesis. Recently a family of ligands for Eph-related receptors has been identified (for a recent review see Pandey et al., 1995a). Up to now, seven ligands have been described (Beckmann et al., 1994, 1995; Bergemann et al., 1995; Cerretti et al., 1995; Cheng and Flanagan, 1994; Davis et al., 1994; Drescher et al., 1995; Kozlosky et al., 1995; Shao et al., 1994, 1995; Winslow et al., 1995). They fall into two structurally distinct classes: five ligands, including B61, Elf1, Rags/Al1, Ehk1-L/Lerk3, and Lerk4, are tethered to the cell surface via a glycosylphosphatidylinositol (GPI) anchor, localized at their hydrophobic C-terminal tail. Two other ligands, Lerk2/Elk-L and Elf2/Htk-L/ Lerk5, have a single hydrophobic transmembrane do- main followed by a long cytoplasmic domain (we will refer to these ligands as Lerk2 and Elf2). Based on observations that the two transmembrane-type ligands specifically interacted with a subset of Eph receptors (the Elk subclass) (Bergemann et al., 1995; Brambilla et al., 1995; Davis et al., 1994) and on similar observations on GPI-anchored ligands interacting with a different set of receptors (Bartley et al., 1994; Cheng and Flanagan, 1994; Davis et al., 1994; Shao et al., 1995; Winslow et al., 1995), we proposed that the Eph receptor family con- sisted of two functionally distinct ligand–receptor sub- 1 Present address: Mount Sinai School of Medicine, Department of Pathology, New York, NY 10029. 2 To whom correspondence should be addressed. Fax: 149-6221 387 516. E-mail: Klein@EMBL-Heidelberg.de. MCN Molecular and Cellular Neuroscience 8, 199–209 (1996) Article No. 0057 199 1044-7431/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.