First printed in R&D Systems' 1999 Catalog.
The process of development involves a series of steps: an initial evagination or invagination, followed by cell proliferation, migration, and differentiation. A number of molecules impact the processes of proliferation and differentiation. Among these are members of the TGF-beta superfamily, the fibroblast growth factor family and various hedgehogs. Cell migration is influenced by “chemodirectants” including a large family of molecules collectively referred to as Ephs. The term Eph derives from the cell line used to identify the first member of the family, EphA1.1 The cell line was an erythropoietin-producing hepatocellular carcinoma cell (thus Eph). The term Eph is now reserved for the tyrosine-kinase members of the family, while the ligands are now referred to as ephrins. The “ephrins” name has two derivations, one from the Greek word efo?os (or ephoros) meaning overseer or controller, and the other from the phrase Eph family receptor interacting proteins.2 For reviews of the Ephs, see references 3-8.
Ephrins are naturally divided into two structural groups.2 The first group, or A class, is composed of GPI (glycosylphosphatidylinositol) linked molecules. The second group, or B class, is composed of transmembrane glycoproteins. The B (transmembrane) type of ephrin will transduce a signal upon binding to an appropriate Eph receptor.9 Apparently, ephrins need to be membrane-bound to activate Ephs, as soluble forms of class A and B ephrins are inactive in Eph phosphorylation assays.10 All ephrins demonstrate four conserved cysteines in their mature segments. Overall, class A ephrins show 23% amino acid (aa) identity in their mature regions,11, 12 while class B ephrins show 33% aa identity in their extracellular segments and 44% aa identity in their cytoplasmic regions.13 In general, class A ephrins bind to class A Ephs, while class B ephrins bind to class B Ephs.3, 6 Unless otherwise stated, all descriptions below are for human ephrins.
Ephrin-A1 is a 25 kDa, 205 aa glycoprotein that has an 18 aa signal sequence and a 187 aa mature segment. The C-terminal 23 aa are believed to participate in GPI-linkage. A1 is inducible on endothelial cells by both TNF-alpha and IL-1 beta 14 and can be found on fetal osteoblasts, odontoblasts, chondrocytes, and squamous epithelium.15 The mature segments of mouse and human ephrin-A1 demonstrate 85% aa identity.15 Ephrin-A1 binding to EphA2 has a Kd = 25 nM.16
Ephrin-A2 is a 213 aa protein that contains a 20 aa signal sequence and a 193 aa mature segment. The mature segment has six cysteines and two potential N-linked glycosylation sites.12 Mouse and human ephrin-A2 are 90% aa identical in the mature segment.12, 17 EphA3 and EphA4 bind to ephrin-A2 with Kds of 1 nM and 10 nM, respectively.17
Ephrin-A3 is a 238 aa polypeptide that contains a 22 aa signal sequence and a 216 aa mature segment. The mature segment shows six cysteines and three potential N-linked glycosylation sites.18 Ephrin-A3 binding to EphA3 has a Kd = 5 nM.18 Ephrin-A3 is noted for its expression in the olfactory system.19
Ephrin-A4 is a 201 aa polypeptide with a 22 aa signal sequence and a 179 aa mature segment.18 The mature protein has one potential N-linked glycosylation site and seven cysteines. Ephrin-A4 binds to EphA4 with a Kd = 5 nM and to EphB1 with a Kd = 20 nM.18 Mouse and human ephrin-A4 show 86% aa identity in the mature segment.12
Ephrin-A5 is a 28 kDa, 228 aa glycoprotein that contains a 20 aa signal sequence and a 208 aa mature segment. The mature segment contains six cysteines and one N-Linked glycosylation site.11, 20 Between mouse and human ephrin-A5, there is 99% aa identity in the mature segment.21 In the mouse, there is also an alternatively spliced short form (a 27 aa deletion) which may be reflected in the human.21 Ephrin-A5 is found on astrocytes and skeletal muscle.
Ephrin-B1 is a 45 kDa, 346 aa glycosylated polypeptide that contains a 24 aa signal sequence, a 211 aa extracellular region, a 26 aa transmembrane (TM) domain and an 83 aa cytoplasmic segment.10, 22 There is 95% aa identity in the extracellular segment of mouse and human ephrin-B1.23 The Kd for ephrin-B1 binding to EphB1 is 925 pM, while the Kd for ephrin-B1 binding to EphA3 is 350 nM, emphasizing the general class specificity of the ephrins.22 A potential proteolytic cleavage site on B1 has been identified.22
Ephrin-B2 is a 38-42 kDa, 333 aa glycoprotein with a 25 aa signal sequence, a 199 aa extracellular region, a 26 aa TM segment and an 83 aa cytoplasmic domain.13, 24 There is 98% aa identity in the extracellular region of mouse and human ephrin-B2.24, 25 Ephrin-B2 is found on bone marrow fibroblasts,26 activated melanocytes and melanoma cells,27 monocytes, mesangial cells and CD34+ stem cells.25 The Kd for ephrin-B2 binding to EphB4 is 535 pM.25
Ephrin-B3 is a 50 kDa, 340 aa glycoprotein that contains a 28 aa signal sequence, a 196 aa extracellular region, a 25 aa TM region and a 91 aa cytoplasmic domain.13, 28 Based on the extracellular region, there is evidence for proteolytic cleavage of this ligand.13 There is 95% aa identity in the extracellular regions of mouse and human ephrin-B3.29 When the extracellular regions for all three B class ligands are aligned, pairwise comparisons demonstrate 50% aa identity for B1 and B2, 42% aa identity for B1 and B3, and 38% aa identity for B2 and B3.29
Ephs are also divided into two classes based on their extracellular structures and specific ligands.2 In general, all the Eph receptors contain an N-terminal Ig-like domain, a cysteine-rich region with 19 conserved cysteines and two fibronectin type III domains. The cytoplasmic region contains a typical tyrosine kinase organization.6, 7 In the A class, EphA1 binds to ephrin-A1, EphA2 through EphA8 bind to ephrins-A1 through -A5, and EphA4 binds to ephrin-B1. In the B class, the Ephs appear to be restricted to B class ephrins.3 All the Ephs listed below are human in origin unless otherwise stated.
EphA1 is a 984 aa type I TM protein with a predicted MW of 109 kDa.1 The molecule has a 23 aa signal sequence, a 524 aa extracellular region, a 21 aa TM segment and a 416 aa cytoplasmic domain.1 A partial mouse clone has been isolated and found to be approximately 80% identical to the human protein.30
EphA2 was first isolated from keratinocytes (thus its alternative name, eck, epithelial cell kinase).31 The molecule is 130 kDa and 976 aa long. It contains a 17 aa signal sequence, a 517 aa extracellular segment, a 24 aa TM region and a 418 aa cytoplasmic domain. EphA2 has also been found in Schwann cells,31 the primitive streak and hindbrain in a very restricted expression pattern.32
EphA3 is a 135 kDa, 983 aa type I TM glycoprotein that contains a 20 aa signal sequence, a 521 aa extracellular region, a 24 aa TM domain and a 418 aa cytoplasmic segment33 The extracellular region has five N-linked glycosylation sites. In the extracellular region, mouse and human EphA3 are 96% identical at the aa level.34 The mouse molecule may generate an alternatively spliced soluble form.34
EphA4 is a 130 kDa, 963 aa TM glycoprotein that consists of a 528 aa extracellular region, a 22 aa TM domain and a 417 aa cytoplasmic segment.35, 36 Although the mouse and human extracellular regions are 98% identical at the aa level, there is a 24 aa addition in the human region.36, 37 Cells that express EphA4 include keratinocytes, B cells and T cells.35, 38
EphA5 is a 1037 aa TM protein that is alternatively known as bsk for brain-specific kinase.36, 39 The protein consists of a 549 aa extracellular region, a 21 aa TM segment and a 443 aa cytoplasmic domain. The mouse and human extracellular regions show 97% aa identity. Mouse EphA5 differs markedly from the human sequence, however, in that it lacks a 164 aa insert. Thus, it contains only 12 cysteine residues and one fibronectin type III domain.39 In humans, there is a cytoplasmic alternate splice variant that contains a deletion of the kinase region.40 The expression of EphA5 appears to be restricted to the brain.36
EphA6 has been identified in the mouse and is a 1035 aa TM protein that contains a 22 aa signal sequence, a 521 aa extracellular region, a 25 aa TM segment and a 467 aa cytoplasmic domain.41 Mouse and rat EphA6 are virtually identical at the aa level with the exception of 87 aa (a C-terminal extension in the mouse molecule. EphA6 is expressed in both adult and fetal cochlear ganglion cells.41
EphA7 is a 998 aa type I TM protein that contains a 24 aa signal sequence, a 532 aa extracellular region, a 21 aa TM domain and a 421 aa cytoplasmic segment.36 It has been found on fetal pro- and pre-B cells.38
Only a partial clone of human EphA8 has been reported.42 The mouse receptor is a 120 kDa, 977 aa type I TM glycoprotein with a 513 aa extracellular region, a 21 aa TM domain and a 443 aa cytoplasmic segment.43 EphA8 is considered specific for GPI-linked ligands and exhibits a Kd of 1.3 nM for ephrin-A2 binding, a Kd of 1.1 nM for ephrin-A3 binding, and a Kd of 500 pM for ephrin-A5 binding.43
EphB1 is a 967 aa TM protein that contains a 523 aa extracellular region, a 20 aa TM domain and a 424 aa cytoplasmic segment. Rat and human EphB1 are 99% identical at the aa level.44 EphB1 is found on endothelial cells and is activated by ephrin-B1, an event that initiates the assembly of endothelial cells into capillary-like cords.45, 46
EphB2 is a 969 aa, type I TM protein that contains a 522 aa extracellular region, a 26 aa TM segment and a 421 aa cytoplasmic domain.47 Mouse and human EphB2 are 99% identical at the aa level.48 EphB2 seems to be transiently expressed on axons only during their outgrowth or migration.
EphB3 is a 130 kDa, 998 aa TM glycoprotein that contains a 33 aa signal sequence, a 523 aa extracellular region, a 26 aa TM domain and a 416 aa cytoplasmic segment.49 In the adult, it is apparently expressed on macrophages. The extracellular regions of mouse and human EphB3 are 96% identical at the aa level.50
EphB4 is a 120 kDa, 972 aa type I TM glycoprotein with a 524 aa extracellular region, a 21 aa TM segment and a 427 aa cytoplasmic domain.51 The extracellular regions of mouse and human are somewhat varied, showing only 88% aa identity.50-52 EphB4 is found on CD34+ stem cells,51 BFU-E26 and secretory mammary epithelium.53
EphB5 has only been reported in the chicken. The molecule is 1000 aa long with a 29 aa signal sequence, 529 aa extracellular domain, 24 aa TM region and 418 aa cytoplasmic segment. Consistent with other ephs, the extracellular region has 19 conserved cysteines and two fibronectin type III domains.54
EphB6 is a 135 kDa type I TM glycoprotein that consists of a 561 aa extracellular region, a 26 aa TM segment and a 403 aa cytoplasmic domain.55 There is 93% aa sequence identity between mouse and human EphB6.56 In both the human and mouse, the kinase domain is inactive. The function of such a receptor is unknown. The non-functionality of the receptor is further complicated by the fact that, in the mouse, there is a possibility of an alternatively spliced secreted form.56
One of the most important functions of Ephs involves cell and axon guidance. In contrast to the chemoattractive agents of immunology, Ephs/ephrins act as chemorepulsive agents. Both Ephs and ephrins can be expressed on axons and cells of neural tube/neural crest derivation. This expression is often of a temporal nature providing repulsive cues for migrating cells and processes.3, 6, 7 For instance, during the migration of neural crest cells and spinal motor neurons, EphB molecules on these cells encounter ephrins expressed on cells of the developing somite. This contact generates a repulsive signal, causing the cells and axons to either retreat or undergo redirection.4-6 In addition, during axon migration in the brain, axons will express B type ephrins, while surrounding neurons or astrocytes express B type Ephs.6, 57 This reverse pattern of expression results in a repulsive cue for migrating axons, emphasizing that ephrin ligands (at least B type) may transduce a functional signal.6 The outcome of such repulsive guidance is a highly complex neural topography that characterizes all aspects of the nervous system.