First printed in R&D Systems' 1998 Catalog.
|Figure 1: TNF Superfamily involving Fas/FasL and CD40/CD40L.|
The first suggestion that a tumor necrotizing molecule existed was made when it was observed that cancer patients occasionally showed spontaneous regression of their tumors following bacterial infections.1 Subsequent studies in the 1960s indicated that host-associated (or endogenous) mediators, manufactured in response to bacterial products, were likely responsible for the observed effects.2, 3 In 1975, it was shown that a bacterially-induced circulating factor had strong anti-tumor activity against tumors implanted in the skin in mice.2, 4 This factor, designated tumor necrosis factor (TNF), was subsequently isolated,5 cloned,6 and found to be the prototype of a family of molecules that are involved with immune regulation and inflammation.2, 7, 8 The receptors for TNF and the other members of the TNF superfamily also constitute a superfamily of related proteins.9-12 Since a number of reviews have been published on the TNF superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF),2, 7-13 this review is designed only to provide simple, basic background information on all of the currently known receptors and ligands in this superfamily.
TNF-related ligands usually share a number of common features. These features do not include a high degree of overall amino acid (aa) sequence homology.7, 9 With the exception of nerve growth factor (NGF) and TNF-beta, all ligands are synthesized as type II transmembrane proteins (extracellular C-terminus) that contain a short cytoplasmic segment (10-80 aa residues) and a relatively long extracellular region (140-215 aa residues).7 NGF, which is structurally unrelated to TNF, is included in this superfamily only because of its ability to bind to the TNFRSF low affinity NGF receptor (LNGFR). NGF has a classic signal sequence peptide and is secreted. TNF-beta, in contrast, although also fully secreted, has a primary structure much more related to type II transmembrane proteins. TNF-beta might be considered as a type II protein with a non-functional, or inefficient, transmembrane segment.7, 8 In general, TNFSF members form trimeric structures, and their monomers are composed of beta-strands that orient themselves into a two sheet structure.8, 10, 11 As a consequence of the trimeric structure of these molecules, it is suggested that the ligands and receptors of the TNSF and TNFRSF superfamilies undergo "clustering" during signal transduction.11, 13
Human NGF is a 12.5 kDa, nonglycosylated polypeptide 120 aa residues long.14, 15 Synthesized as a prepropeptide, there is an 18 aa residue signal sequence, a 103 aa residue N-terminal pro-sequence, and a 120 aa residue mature segment. Human to mouse, there is 90% aa sequence identity in the mature segment. In the mouse, NGF is referred to as beta-NGF, due to the existence of NGF in a 130 kDa (7S) heterotrimeric (alpha beta gamma) complex in submaxillary glands.15, 16 Many cells, however, do not synthesize all the components of this 7S complex, and the typical form for NGF is a 25 kDa, non-disulfide linked homodimer.14, 16 NGF and all other neurotrophins bind to the LNGFR, a member of the TNFRSF.17
Human CD40L is a 39 kDa, type II (extracellular C-terminus) transmembrane glycoprotein that was originally identified on the surface of CD4+ T cells.18 With a predicted molecular weight of 29 kDa, CD40L is 261 aa residues long, with a 22 aa residue cytoplasmic domain, a 24 aa residue transmembrane segment, and a 215 aa residue extracellular region.18 Human to mouse, CD40L is 73% identical at the aa sequence level and mouse CD40L is apparently active in humans.19 Although usually considered to be a membrane bound protein, natural, proteolytically cleaved 15-18 kDa soluble forms of CD40L with full biological activity have also been described.20, 21 Like TNF-alpha, CD40L is reported to form natural trimeric structures.20, 22 Cells known to express CD40L include B cells, CD4+ and CD8+ T cells,23 mast cells and basophils,24 eosinophils,25 dendritic cells,26 and monocytes, NK cells, and gd T cells.27
Mouse 4-1BBL is a 50 kDa, 309 aa residue transmembrane glycoprotein that is the largest of the TNFSF members.28 With a predicted molecular weight of 34 kDa, the molecule has an 82 aa residue cytoplasmic region, a 21 aa residue transmembrane segment, and a 206 aa residue extracellular domain. Although human and mouse 4-1BB molecules exhibit 60% identity at the aa level, human and mouse 4-1BBL molecules exhibit only 36% identity at the aa level. This level of cross species conservation is much lower than that shown by other members of the TNFSF.11, 29 In mice, two ligands are known for 4-1BB: 4-1BBL and laminin.30 Cells known to express 4-1BBL include B cells, dendritic cells, and macrophages.31, 32
Human TNF-alpha is a 233 aa residue, nonglycosylated polypeptide that exists as either a transmembrane or soluble protein.6, 33, 34 When expressed as a 26 kDa membrane bound protein, TNF-alpha consists of a 29 aa residue cytoplasmic domain, a 28 aa residue transmembrane segment, and a 176 aa residue extracellular region.7, 33 The soluble protein is created by a proteolytic cleavage event via an 85 kDa TNF-alpha converting enzyme (TACE),35, 36 which generates a 17 kDa, 157 aa residue molecule that normally circulates as a homotrimer.6, 37, 38 Normal levels of circulating TNF are reported to be in the 10-80 pg/mL range.39, 40 While both membrane-bound and soluble TNF-alpha are biologically active, soluble TNF-alpha is reported to be more potent.41 Mouse to human, full-length TNF-alpha shows 79% aa sequence identity.42, 43 Unlike human TNF-alpha, mouse TNF-alpha is glycosylated.42, 43 The variety of cell types known to express TNF-alpha is enormous and includes macrophages, CD4+ and CD8+ T cells,44 adipocytes,45 keratinocytes,46 mammary and colon epithelium,47, 48 osteoblasts,49 mast cells,50 dendritic cells,51 pancreatic beta-cells,52 astrocytes,53 neurons,54 monocytes,55 and steroid-producing cells of the adrenal zona reticularis.56
OX40, the receptor for OX40L, is a T cell activation marker with limited expression that seems to promote the survival (and perhaps prolong the immune response) of CD4+ T cells at sites of inflammation.57 OX40L also shows limited expression. Currently only activated CD4+, CD8+ T cells,58 B cells,59, 60 and vascular endothelial cells have been reported to express this factor.61 The human ligand is a 32 kDa, 183 aa residue glycosylated polypeptide that consists of a 21 aa residue cytoplasmic domain, a 23 aa residue transmembrane segment, and a 139 aa residue extracellular region.7, 57 When compared to the extracellular region of TNF-alpha, OX40L has only 15% aa sequence identity, again emphasizing the importance of secondary and tertiary structures as the basis for inclusion in the TNF Superfamily.57 Human OX40L is 46% identical to mouse OX40L at the aa sequence level. Mouse OX40L is active in humans, but human OX40L is inactive in mice.58 Consistent with other TNFSF members, OX40L is reported to exist as a trimer.62
Human CD27L is a 50 kDa, 193 aa residue type II (extracellular C-terminus) transmembrane glycoprotein that appears to have a very limited immune system expression pattern.63, 64 Having less than 25% aa sequence identity to TNF-alpha and CD40L, the molecule has only a 20 aa residue cytoplasmic segment, an 18 aa residue transmembrane domain, and a 155 aa residue extracellular region.64 Although the 20 aa residue cytoplasmic segment is short by most standards, there is a suggestion that it has a signaling function, perhaps activating the cytolytic program of gd T cells65 and/or contributing necessary signals for antibody production in B cells.66 Cells known to express CD27L are usually activated cells and include NK cells,67 B cells,66 CD45RO+, CD4+ and CD8+ T cells,68 gd T cells,65 and certain types of leukemic B cells.69
Fas ligand (FasL) is a highly conserved, 40 kDa transmembrane glycoprotein that occurs as either a membrane bound protein or a circulating homotrimer.70, 71 In humans, FasL is synthesized as a 281 aa residue protein with an 80 aa residue cytoplasmic region, a 22 aa residue transmembrane segment, and a 179 aa residue extracellular domain.70 When proteolytically cleaved, FasL is a 70 kDa homotrimer composed of 26 kDa monomers with full biological activity.71 In mice, the FasL is somewhat different. Although mouse FasL molecule has 77% aa sequence identity with human FasL,70, 72, 73 polymorphisms exist in the mouse FasL, leading to functionally distinct FasL forms.74 In addition, a one aa residue substitution at position 273 (Phe to Leu) results in the gld/gld (generalized lymphoproliferative disease) mutation.72 Finally, while FasL in a membrane-bound form shows species cross-reactivity,70 soluble mouse FasL is apparently biologically inactive.71 Cells known to express FasL include type II pneumocytes and bronchial epithelium,75 monocytes,76 LAK cells and NK cells,77, 78 dendritic cells,79 B cells,80 macrophages,81 CD4+ and CD8+ T cells,82 and colon and lung carcinoma cells.75, 83
Human CD30L is a 40 kDa, 234 aa residue transmembrane glycoprotein with 72% aa sequence identity to its mouse counterpart.84 With a predicted molecular weight of 26 kDa, the molecule consists of a 46 aa residue cytoplasmic region, a 21 aa residue transmembrane segment, and a 172 aa residue extracellular domain.84 Species cross-reactivity has been reported.84 As suggested for CD27L, the cytoplasmic region is suggested to transduce a signal.85 The CD30/CD30L system is complex since CD30 ligation can induce both proliferation and apoptosis.84 Cells known to express CD30L include monocytes and macrophages,84 B cells plus activated CD4+ and CD8+ T cells,86 neutrophils, megakaryocytes, resting CD2+ T cells, erythroid precursors,87 and eosinophils.88
TNF-beta, otherwise known as lymphotoxin-alpha (LT-alpha) is a molecule whose cloning was contemporary with that of TNF-alpha.89 Although TNF-beta circulates as a 171 aa residue, 25 kDa glycosylated polypeptide, a larger form has been found that is 194 aa residues long.90 The human TNF-beta cDNA codes for an open reading frame of 205 aa residues (202 in the mouse),89, 91 and presumably some type of proteolytic processing occurs during secretion. As with TNF-alpha, circulating TNF-beta exists as a non-covalently linked trimer and is known to bind to the same receptors as TNF-alpha.92-95 Circulating TNF-beta levels are reported to be about 150 pg/mL.96 Human TNF-beta is 72% identical to mouse TNF-beta at the aa sequence level across the entire molecule.91 TNF-alpha to TNF-beta, aa sequence identity is reported to be 28%.6, 93 Unlike TNF-alpha, TNF-beta does not have a transmembrane form. However, it can be membrane-associated, due to its binding to membrane-anchored LT-beta (see below).92, 97 In this complex, TNF-beta and LT-beta will form a heterotrimer that binds to both the LT-beta receptor and TNFRI receptor. Activation of the TNFRI receptor, however, does not occur.92, 94 Cells known to express TNF-beta include NK cells, T cells and B cells.97
Human lymphotoxin-beta (LT-beta), also known as p33, is a 33 kDa type II (extracellular C-terminus) transmembrane glycoprotein originally cloned from a T cell hybridoma cell line. It is 244 aa residues long, and has a 16 aa residue cytoplasmic segment, a 31 aa residue transmembrane domain, and a 197 aa residue extracellular region.7, 98 On the membrane surface, LT-beta readily forms a trimeric complex with TNF-beta, in either a 2:1 (major form) or a 1:2 (minor form) ratio.92, 98 LT-beta is not secreted.94 A comparison of human to mouse LT-beta shows 80% aa sequence identity in homologous regions.99 Overall, however, the mouse gene shows significant differences from the human gene. In mice, an intron has been incorporated into the genome creating a 66 aa residue insert into what would otherwise be a 240 aa residue molecule.100
TRAIL, or TNF-related apoptosis-inducing ligand, is a newly discovered TNFSF member initially cloned from human heart and lymphocyte cDNA libraries.101 With a predicted molecular weight of 32 kDa, human TRAIL is 281 aa residues long, with a 17 aa residue cytoplasmic tail, a 21 aa residue transmembrane segment, and 243 aa residue extracellular region.101, 102 Human TRAIL is 65% identical to mouse TRAIL at the aa sequence level across the entire molecule and there is complete species cross-reactivity.101 As a membrane bound protein, TRAIL shows a trimeric structure.102 Although TRAIL is known to be expressed by lymphocytes, many tissues seem to express the ligand, and this broad expression pattern suggests an intriguing function for the molecule.101
As with members of the TNF Superfamily, members of the TNF Receptor Superfamily (TNFRSF) also share a number of common features. In particular, molecules in the TNFRSF are all type I (N-terminus extracellular) transmembrane glycoproteins that contain one to six ligand-binding, 40 aa residue cysteine-rich motifs in their extracellular domain.7, 9-11 In addition, functional TNFRSF members are usually trimeric or multimeric complexes that are stabilized by intracysteine disulfide bonds. Unlike most members of the TNFSF, TNFRSF members exist in both membrane-bound and soluble forms.9 Finally, although aa sequence homology in the cytoplasmic domains of the superfamily members does not exceed 25%,7 a number of receptors are able to transduce apoptotic signals in a variety of cells, suggesting a common function.9, 103
The human low-affinity nerve growth factor receptor (LNGFR) is a 75 kDa, 427 aa residue type I (extracellular N-terminus) transmembrane glycoprotein. The 427 aa residue receptor contains a 25 aa residue signal sequence, a 225 extracellular region, a 23 aa residue transmembrane segment, and a 154 aa residue cytoplasmic domain.7, 104, 105 There are four cysteine-rich domains in its extracellular region. A comparison of human to rat LNGFR shows 92% aa sequence identity in the extracellular domain, and 95% aa sequence identity in the cytoplasmic region.104, 106 In its functional form, it often appears as an approximately 200 kDa disulfide-linked homodimer.104, 105 All neurotrophins bind to LNGFR with the same Kd of approximately 1-3 nM.17, 105, 106 In contrast to the high-affinity neurotrophin receptors (Trks), LNGFR has no inherent tyrosine kinase activity.107 It has been suggested that LNGFR passes NGF to the physiologically-active Trks.108, 109 However, recent evidence now suggests that co-expressed LNGFR and TrkA modulate each others activities110, 111 and that LNGFR signals on its own, utilizing a functional "death domain" in its cytoplasmic region.112, 113 Soluble forms of 35- 45 kDa LNGFR are known to occur, presumably the result of proteolytic cleavage.114 Cells known to express LNGFR include oligodendrocytes,113 B cells (but not monocytes or T cells),115 bone marrow fibroblasts,116 autonomic and sensory neurons,110, 117 Schwann cells,117 follicular dendritic cells,118 select astrocytes,119 and mesenchymal cells involved with mesenchymal-epithelial interactions.120
CD40 is a 50 kDa, 277 aa residue transmembrane glycoprotein most often associated with B cell proliferation and differentiation.121, 122 Expressed on a variety of cell types, human CD40 cDNA encodes a 20 aa residue signal sequence, a 173 aa residue extracellular region, a 22 aa residue transmembrane segment, and a 62 aa residue cytoplasmic domain.122 There are four cysteine-rich motifs in the extracellular region that are accompanied by a juxtamembrane sequence rich in serines and threonines. Mouse CD40 is 62% identical to human CD40 at the aa sequence level. However, mouse CD40 is 305 aa residues long with the difference attributable to a 28 aa residue extension in the cytoplasmic tail.123 CD40 ligation is associated with the induction of apoptosis. This is not due the activation of a cytoplasmic "death domain"; rather CD40 ligation can upregulate Fas antigen, which primes cells for subsequent Fas-mediated apoptosis.124 Currently, it is believed that the normal signaling pathway of CD40 involves both NF-kB, and protein kinase (lyn) activation.125 Soluble CD40 has been identified in B cell cultures, presumably the result of proteolytic processing.126, 127 Although many functions have been attributed to CD40, one suggests that CD40 ligation preferentially drives B cells into memory cells rather than plasma cells.128 Cells known to express CD40 include B cells.123 monocytes and basophils (but not mast cells),129 eosinophils,130 endothelial cells,131 interdigitating dendritic cells,132 Langerhans cells,133 blood dendritic cells,134 fibroblasts,135 keratinocytes,136 Reed-Sternberg cells of Hodgkin's disease, and Kaposi's sarcoma cells.137, 138 A review on CD40 can be found in reference 121.
Human CD137 is a 30-35 kDa activation-induced glycoprotein that occurs as both a monomer and homodimer on the surface of cells.7, 139-141 CD137 is aa residues long, including a 17 aa residue signal sequence, a 169 aa residue extracellular region, a 27 aa residue transmembrane segment, and a 42 aa residue cytoplasmic domain.29, 139, 142 In the extracellular region, CD137 contains the characteristic multiple cysteine-rich motif.7 Mouse to human, although there is 60% aa sequence identity across the open reading frame,29, 143 there is minimal to no cross-species biological activity.29, 144 The Kd for CD137L binding to CD137 is reported to be about 30 pM.29 Soluble CD137 is known to exist, but unlike the soluble forms of TNFRI & II, CD40 and LNGFR, it is created by an alternative splicing event.145 CD137 ligation is reported to interrupt T cell apoptotic programs associated with activation-induced cell death.146 Cells known to express CD137/4-1BB/ILA (for induced by lymphocyte activation) include fibroblasts,145 thymocytes,145 monocytes,139, 145 and CD4+ and CD8+ T cells.141
TNFRI is a 55 kDa, 455 aa residue transmembrane glycoprotein that is apparently expressed by virtually all nucleated mammalian cells.147-149 The molecule has a 190 aa residue extracellular region, a 25 aa residue transmembrane segment, and a 220 aa residue cytoplasmic domain.7, 147 In a comparison of mouse to human proteins, TNFRI has 64% aa sequence identity (70% in the extracellular region), with mouse and human TNFRI binding human and mouse TNF-alpha with equal affinity.150, 151 The extracellular region has four cysteine-rich motifs, the first of which is suggested to be required for binding.152 The cytoplasmic domain has an 80 aa residue "death domain" that can trigger an apoptotic pathway.153 This is not the only outcome of TNFRI ligation, however. NF-kB is also activated by the TNFRI, although the mechanism determining the choice of pathways is not clear.154 Both TNF-alpha and TNF-beta bind to TNFRI. Soluble TNF-alpha binds with a Kd in the range of 20-60 pM,152, 154 while TNF-beta binds with a Kd equal to 650 pM.152 While TNFRI relative to TNFRII has been suggested to be the more physiologically-relevant receptor, recent evidence suggests that TNFRI is most important for circulating TNF-alpha, while membrane-bound TNF-alpha associates with TNFRII154 (see TNFRII below). Soluble TNFRI, which blocks TNF-alpha activity, has been identified in both urine and blood (1-3 ng/mL).39, 40, 155 Soluble forms of at least two molecular weights (32 kDa and 48 kDa) have been identified and are believed to be generated by proteolytic cleavage.149, 156, 157 Among the numerous cells known to express TNFRI are hepatocytes,40 monocytes and neutrophils,158 cardiac muscle cells,159 endothelial cells,160 and CD34+ hematopoietic progenitors.161
Human TNFRII is a 75 kDa, 461 aa residue transmembrane glycoprotein originally isolated from a human lung fibroblast library.162 This receptor consists of a 240 aa residue extracellular region, a 27 aa residue transmembrane segment and a 173 aa residue cytoplasmic domain.7, 162 Mouse to human, aa sequence identity in TNFRII cytoplasmic domain is 73 %, while aa sequence identity in the extracellular region falls to 58%.150 This drop in extracellular identity is reflected in the observation that human TNF-alpha is not active in the mouse system.150 TNFRII to TNFRI, aa sequence identity is only about 20% in the extracellular region and 5% in the cytoplasmic domain.150 The function of TNFRII is not clear. In the TNF-alpha system, it has been suggested that TNFRII binds TNF-alpha and transfers it to TNFRI, which then is activated and initiates a physiological response.163, 164 TNF-alpha binding to TNFRII clearly has an effect on cells, however, inducing apoptosis in rhabdomyosarcoma (skeletal muscle tumor) cells,165 and cell migration in Langerhans cells.166 A clue to understanding of TNFRII activity may lie in its binding kinetics. At 37 °C, soluble TNF-alpha binds to TNFRI with a Kd of 20 pM, and to TNFRII with a Kd of 300 pM (note: at 4 °C the Kd's are approximately equal at 100 and 300 pM respectively). Since TNF-alpha levels (at least systemically) are usually in the range of 100 pM, TNF-alpha activity will normally be mediated by the TNFRI molecule. In addition, a TNF-alpha:TNFRII interaction leads to a very slow oligomerization of receptor molecules, and ligand dissociation seems to occur before receptor-signaling complex formation. Thus, TNFRII could be envisioned to "hand-off" to TNFRI. However, not all TNF-alpha is soluble, and current theory predicts that membrane-bound TNF-alpha is the effective ligand for TNFRII. In this form, a TNF-alpha:TNFRII complex allows time for the slow formation of signal-transducting oligomers.154 For TNF-beta, the Kd for TNFRII binding is reported to be approximately 300 pM. However, it would appear that a TNF-beta:TNFRII complex is non-signaling, leading to the suggestion that in the TNF-beta system, TNFRII is nothing more that a "decoy-receptor".167 Soluble forms of TNFRII have been identified, resulting apparently from proteolytic cleavage by a metalloproteinase termed TRRE (TNF-Receptor Releasing Enzyme).168, 169 The shedding process appears to be independent of that for soluble TNFRI.170 Among the multitude of cells known to express TNFRII are monocytes,170 endothelial cells,171 Langerhans cells,166 and macrophages.172
Human OX40 is a 48 kDa, type I (external N-terminus) transmembrane glycoprotein that appears to have a very limited pattern of expression,173, 174 currently consisting of only activated CD4+ and CD8+ T cells.174 The mature molecule is a 250 aa residue polypeptide that consists of a 188 aa residue extracellular region, a 26 aa residue transmembrane segment, and a 36 aa residue cytoplasmic domain.7, 173 In the extracellular region, there is about 60% aa sequence identity human to mouse.173, 175 There is marked species cross-reactivity in this system.58, 174
Immune system cells are currently the only reported source for expression of CD27, a 50-55 kDa variably glycosylated polypeptide.176, 177 The mature molecule has a predicted molecular weight of 27 kDa and is 242 aa residues long, consisting of a 175 aa residue extracellular region, a 21 aa residue transmembrane segment, and a 46 aa residue cytoplasmic domain.7, 176 Mouse to human, CD27 is 65% identical at the aa sequence level, with both molecules expressed as homodimers on the cell surface.176, 178 Although CD27 lacks a recognizable cytoplasmic "death-domain" motif, it can induce apoptosis through a receptor-associated, death-domain containing a cytoplasmic protein known as Siva (the Hindu god of destruction).179 Whether there are a number of such proteins specific for various TNFRSF members remains to be seen. A soluble, 32 kDa form of CD27 has been identified in both blood and urine, most likely the result of proteolytic processing.177, 180 Cells known to express CD27 include NK cells,181 B cells,182, 183 CD4+, CD8+ T cells and thymocytes.176
Human Fas (fibroblast associated) is a 43 kDa, 355 aa residue transmembrane glycoprotein found on multiple cell types.184 Also known as APO-1 (for Apoptosis-1), the molecule appears to be more than a simple mediator of apoptosis. On fibroblasts, Fas ligation can lead to either proliferation or apoptosis depending on the relative number of expressed Fas molecules.185 The human receptor is 335 aa residues long, with a 156 aa residue extracellular region, a 20 aa residue transmembrane segment, and a 144 aa residue cytoplasmic domain.7, 184 In the extracellular region, there are three cysteine-rich motifs, while in the cytoplasmic region there is a 68 aa residue "death-domain", which is also found in (and 25% identical to) the TNFRI cytoplasmic region.153, 186 It is currently suggested that cytoplasmic death-domain containing proteins associate with this area (FADD protein with Fas, TRADD protein with TNFRI), thereby transmitting apoptotic signals.187 Both FADD and TRADD are also known to associate with each other, suggesting considerable interaction between the apoptotic programs of each system.187 There is 50% aa sequence identity in Fas molecules, mouse to human, with mouse Fas being eight aa residues shorter in length.188 Soluble forms of Fas are known, the result of alternative gene splicing.189, 190 In blood, soluble Fas is reported to circulate as a dimer and trimer at low ng/mL concentrations.190 Cells reported to express Fas include CD34+ stem cells,161 fibroblasts,185 NK cells,191 keratinocytes,92 hepatocytes,193 B cells and B cell precursors,194 monocytes plus CD4+ and CD8+ T cells,195 CD45RO+ gamma deltaT cells,196 eosinophils,197 and thymocytes, with low levels detected on CD4-CD8- precursors, and high levels on CD4+CD8+ precursors.198 A review on Fas can be found in reference #199.
Human CD30 is a 105-120 kDa transmembrane glycoprotein often associated with the Reed-Sternberg cells of Hodgkin's disease.200, 201 Although in most cases, mouse to human, members of the TNFRSF are close in terms of overall length, CD30 shows a marked departure from the norm. Mature human CD30 is 577 aa residues long, with an 18 aa residue signal sequence, a 365 aa residue extracellular region, a 24 aa residue transmembrane segment, and a 188 aa residue cytoplasmic domain.200 There are six cysteine-rich motifs in the extracellular region. In mice, mature CD30 is 480 aa residues long, with a 90 aa residue deletion in the extracellular region relative to the human.202 This 90 aa residue differential eliminates three of the six cysteine-rich motifs found in humans.202 Overall, there is approximately 60% aa sequence identity, mouse to human.202 An 85 kDa form of soluble CD30 has been detected in the blood of patients with CD30+ lymphomas.203 Cells known to express CD30 include Reed-Sternberg cells,201 CD8+ T cells,202 and CD4+ T cells.204 Of note, CD30+ CD4+ T cells are considered to be major producers of T cell-derived IL-5.204
Human LT-beta R (lymphotoxin-beta receptor) is a 75 kDa transmembrane glycoprotein that consists of a 201 aa residue extracellular region, a 26 aa residue transmembrane segment, and a 187 aa residue cytoplasmic domain.7, 205, 206 In the extracellular region, it contains four cysteine-rich motifs. A comparison of mouse to human receptors shows 76% identity at the aa sequence level.206 In terms of ligands, LT-beta R preferentially binds (TNF-beta)1(LT-beta)2 heterotrimers over LT-beta homotrimers. Mouse ligands are active on human receptors while human ligands are only marginally active on mouse receptors.206 Relative to the TNFR receptors, LT-beta R is most like TNFRI in the first two cysteine-rich motifs, and most like TNFRII in the third and fourth cysteine-rich motifs.206 LT-beta R is known both to activate NF-kappaB and to induce cell death via TRAF-3, making it somewhat analogous to TNFRI.207 Genes known to be activated by LT-beta R include IL-8 and RANTES.208 Based on cell lines, LT-beta R is found on monocytes, fibroblasts, smooth muscle and skeletal muscle cells.208
DR3 (or Death Receptor 3) is a 54 kDa, 417 aa residue type I (external N-terminus) transmembrane glycoprotein that has been isolated under a variety of names.209 The DR3 designation results from this being the third factor discovered with an intracellular "death domain", TNFRI being the first and Fas being the second.209 Also known as APO-3,210 Wsl-1,211 LARD (lymphocyte-associated receptor of death),212 and TRAMP (TNFR-related apoptosis mediating protein),213 this molecule appears to be somewhat analogous to TNFRI in that it can activate both NF-kappaB and induce apoptosis.209, 213 The receptor has a 24 aa residue signal sequence, a 178 aa residue extracellular region, a 23 aa residue transmembrane segment, and a 192 aa residue cytoplasmic domain.209, 210 In the extracellular region there are four cysteine-rich motifs.210 At the aa sequence level, DR3 is approximately 30% identical to TNFRI, and 25% identical to Fas.210 About a dozen alternate splice forms are known for DR3, many coding for potentially soluble forms.211-213 The shorter isoforms seem to be expressed by resting cells that subsequently switch to expressing the full-length (413 aa residues) isoform upon activation.212 Cells identified as expressing DR3 include T and B cells212 and HUVECs (human umbilical vein endothelial cells). A HUVEC library was used to clone DR3.209 There is currently no known ligand for DR3.
DR4 (or Death Receptor 4) is one of three known receptors for TRAIL.214 DR4 is a 468 aa residue type I (extracellular N-terminus) transmembrane protein that contains a 23 aa residue signal sequence, a 226 aa residue extracellular region, a 19 aa residue transmembrane segment, and a 220 aa residue cytoplasmic domain. In the extracellular region, there are two cysteine-rich motifs.214 Although DR4 has a death-domain, it cannot activate NF-kappaB, and it cannot use FADD, a death domain-associated cytoplasmic protein utilized by Fas, TNFRI and DR3.214 To date, it is only known to be expressed by activated T cells.214
DR5 (or Death Receptor 5) is the second of three known receptors for TRAIL.215 Like DR4, ligation of this receptor can trigger an apoptotic program independent of FADD participation. The molecule is 411 aa residues long, with a very large 51 aa residue signal sequence, a 132 aa residue extracellular region, a 22 aa residue transmembrane segment, and a 206 aa residue cytoplasmic domain. The extracellular region contains two cysteine-rich motifs.215
DcR1 (Decoy Receptor-1)216 or TRID (TRAIL Receptor without an Intracellular Domain)215 is exactly what the latter name suggests, i.e., a membrane-bound receptor for TRAIL that possesses no cytoplasmic domain. Found on endothelial cells and lymphocytes, the molecule is 259 aa residues long, possessing a 23 aa residue signal sequence, a 217 aa residue extracellular region, and a 19 aa residue transmembrane domain.215 There are two cysteine-rich motifs in the extracellular region, which is 50-60% identical at the aa sequence level to the same regions in DR4 and DR5. Without a cytoplasmic segment, this receptor does not signal. Instead, it inhibits responsiveness to TRAIL at the level of the cell membrane.
TR2 is a newly discovered, 32 kDa type I transmembrane glycoprotein that has no known ligand at present.217 Found on T cells, B cells, monocytes and endothelium, the molecule is 283 aa residues long, with a 36 aa residue signal sequence, a 165 aa residue extracellular region, a 23 aa residue transmembrane segment, and a 59 aa residue cytoplasmic domain. The extracellular region contains four cysteine-rich motifs.217
GITR (glucocorticoid-induced TNFR family-related) is a 228 aa residue transmembrane protein that is suggested to be a close relative of 4-1BB and CD27. Inducible during T cell activation, the molecule has a 19 aa residue signal sequence, a 134 aa residue extracellular region, a 23 aa residue transmembrane segment and a 52 aa residue cytoplasmic domain. It has three cysteine-rich motifs in its extracellular region. Like 4-1BB, ligation interrupts TCR-DC3-induced apoptosis in T cells.218
Named because of its ability to protect bone from breakdown (i.e., inhibit osteoclasts), OPG is a 55 kDa, 380 aa residue, naturally secreted member of the TNFRSF.219 Most similar to TNFRII and CD40, this "receptor" has no transmembrane segment, and circulates as a disulfide-linked homodimer. The human, mouse and rat proteins are all equal in length, with human and rat having 94% aa sequence identity. It is unknown what type of "ligand" exists for this receptor.