Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1

Abstract

Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-speci®c phospholipase C. Using Scatchard analysis, high a nity binding of Elf-1 (1.7610 710 M) and B61 (2.2610 710 M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable a nity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower a nities were observed for the two truncated receptors MDK1-T1 and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These ®ndings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.

Ephrin-B3, a ligand for the receptor EphB3, expressed at the midline of the developing neural tube

“…Nevertheless, the distinctive expression patterns of the receptors soon suggested interesting roles in cellcell signaling. Almost all the receptors are expressed prominently in the developing or adult nervous system, suggesting roles in neural development or function (Becker et al, 1994;Cheng and Flanagan, 1994a;Ciossek et al, 1995;Ganju et al, 1994;Henkemeyer et al, 1994;Lai and Lemke, 1991;Maisonpierre et al, 1993;Nieto et al, 1992;Pasquale et al, 1992;Ruiz and Robertson, 1994;Sajjadi and Pasquale, 1993;Soans et al, 1994;Tuzi and Gullick, 1994;Zhou et al, 1994). Also, many of the receptors are expressed in highly restricted patterns at the stage of gastrulation and organogenesis, suggesting functions in patterning the embryo.…”

The Eek receptor, a member of the Eph family of tyrosine protein kinases, can be activated by three different Eph family ligands

“…More than thirteen distinct members of this receptor subfamily have been reported to date, establishing the Eph family as the largest group among the receptor tyrosine kinase subfamilies (Tuzi and Gullick, 1994;Pandey et al, 1995a;Friedman and O'Leary, 1996). The UPGMA tree by alignment of the Eek amino acid sequence with the sequences of other full-length Eph family members, shows that the Eek protein shares greater overall similarity to some members, including Ehk-3/MDK1 (Ciossek et al, 1995;Valenzuela et al, 1995), Sek/Cek8 (GilardiHebenstreit et al, 1992;Nieto et al, 1992), Ehk-2 (Maisonpierre et al, 1993), Hek/Mek4/Cek4 (Sajjadi et al, 1991;Sajjadi and Pasquale, 1993) and Bsk/ Ehk1/Rek7/Cek7 (Maisonpierre et al, 1993;Zhou et al, 1994). Interestingly, these receptors closely related to Eek are predominantly expressed in the nervous system.…”

“…We would infer from this tree that the Eek protein is closest related to a branch of the family that includes Ehk-3/MDK1 (Valenzuela et al, 1995;Ciossek et al, 1995), Sek/ Cek8 , Ehk-2 (Maisonpierre et al, 1993), Hek/Mek4/Cek4 (Sajjadi et al, 1991) and Bsk/Ehk1/Rek7/Cek7 (Zhou et al, 1994).…”

“…The Figure 2 Cladogram for the Eph subfamily of receptor tyrosine kinases. The whole amino acid sequence of Eek was compared with those of Ehk-2 (Maisonpierre et al, 1993), Hek/Mek4/Cek4 (Sajjadi et al, 1991), Bsk/Ehk1/Rek7/Cek7 (Zhou et al, 1994), Ehk-3/MDK1 (Valenzuela et al, 1995;Ciossek et al, 1995), Sek/ Cek8 (Gilardi-Hebenstreit et al, 1992), Elk/Cek6 (Letwin et al, 1988;Lhotak et al, 1991), Cek5/Erk/Nuk (Henkemeyer et al, 1994), Hek2/Sek4/Cek10 (Bohme et al, 1993), Myk1 (Andres et al, 1994), Eck/Sek2 (Lindberg and Hunter, 1990), and Eph (Hirai et al, 1987) using the PILEUP program (GCG). The 42 amino acid insert, present between kinase motifs IV and V in the Ehk-2 kinase domain was deleted for comparison as previously described (Maisonpierre et al, 1993).…”

“…Other members (e.g. Eph, Hirai et al, 1987;Maru et al, 1988;Eck/Sek2, Lindberg and Hunter, 1990; Cek5/Erk/Nuk, Pasquale, 1991, Pasquale et al, 1992; Hek2/Sek4/Cek10, Bohme et al, 1993;Htk/Myk1, Bennett et al, 1994;Ehk-3/MDK1, Valenzuela et al, 1995;Ciossek et al, 1995) are expressed in a broader range of tissues including lung, kidney, and intestine, as well as brain. Studies involving functional roles for the Eph-related receptors expressed in the developing brain or other tissues have been di cult until the recent ®ndings of their ligands.…”