The EPH-like branch of receptor tyrosine kinases (RTKs) 1 has more than 28 members described in several vertebrate species (1-3). All were identified prior to the characterization of their cognate ligands by methods independent of a biological assays or specific physiological activity (2). As a consequence little is known about their specific functions. However, expression patterns of several EPH-like RTKs in embryogenesis, in particular in the nervous system, suggests a role in development (4, 5). Overexpression of some family members including HEK, EPH, ERK, and ECK in tumor-derived cell lines, tumor specimens, and transfected cells implicate these receptors in oncogenesis (6 -10).Recently we identified HEK on the cell surface of a pre-B acute lymphoblastic leukemia cell line, LK63, using the IIIA4 monoclonal antibody (mAb) (7). Immunofluorescence studies with IIIA4 revealed expression of HEK in blood samples from patients with acute leukemia, but not in normal adult tissues or blood cells (7, 11). In embryos, the expression patterns of the murine and chicken HEK homologues MEK 4 and CEK 4, and their recently identified ligand ELF1 (12) and RAGS (13), respectively, suggest a role in the development of the retinotectal projection map. We isolated a soluble HEK ligand from human placenta-conditioned medium using a biosensor-based affinity detection approach (14). The HEK ligand was identified by sequence homology as a soluble form of AL-1 (15), a member of the ligands for EPH-Related Kinases (LERKs) family (16,17) and for consistency with other members will be referred to as LERK 7. This family of transmembrane or membrane-associated proteins were isolated as potential ligands for EPH-like RTKs through their interactions with recombinant EPH receptor family exodomains (15, 18 -21). In most cases, bivalent Fc fusion proteins of either the receptor or the ligand were used for detecting potential binding partners. A requirement for membrane association of the ligand and the ability of more than one ligand to bind the same receptor with comparable affinity appeared to be characteristic of many of these ligands (21-25). However, soluble ligands for both ECK (26) and HEK (14) have been isolated from biological sources using receptor affinitybased protocols. Functional assays with the natural ligands leave some ambiguity about the absolute requirement for membrane association of the ligands for biological function (9,15,26,27). In addition to binding LERK7, a bivalent HEK fusion construct was shown to bind with significant affinity to LERK 1, 2 (18), LERK 3, 4 (28), and LERK 5 (24).Here we report the results of studies on the interaction between HEK and bivalent Fc-fusion proteins of LERKs 1 to 5 and LERK 7 or monovalent LERK3 and LERK7 produced as Flag-epitope tagged proteins. We use BIAcore technology, SE-HPLC, sedimentation equilibrium centrifugation, and functional assays which show that LERK 7 is the principal HEK
Fc gamma receptors bind IgG to initiate cellular responses against pathogens and soluble antigens. We have determined the three-dimensional structure of the extracellular portion of human Fc gammaRIIa to 2.0 A resolution providing a structural basis for the unique functions of the leukocyte FcR family. The receptor is composed of two immunoglobulin domains and arranged to expose the ligand-binding site at one end of domain 2. Using alanine mutants we find that the binding sites for IgG1 and 2 are similar but the relative importance of specific regions on the receptor varies. In crystals, Fc gammaRIIa molecules associate to resemble V(L)V(H) dimers, suggesting that two Fc gammaRIIa molecules could cooperate to bind IgG in an asymmetric manner.
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