Non-catalytic tyrosine-phosphorylated receptor

Non-catalytic tyrosine-phosphorylated receptors (NTRs), also called immunoreceptors or Src-family kinase-dependent receptors, are a group of cell surface receptors expressed by leukocytes that are important for cell migration and the recognition of abnormal cells or structures and the initiation of an immune response.^[1][2] These transmembrane receptors are not grouped into the NTR family based on sequence homology, but because they share a conserved signalling pathway utilizing the same signalling motifs.^[1] A signaling cascade is initiated when the receptors bind their respective ligand resulting in cell activation. For that tyrosine residues in the cytoplasmic tail of the receptors have to be phosphorylated, hence the receptors are referred to as tyrosine-phosphorylated receptors. They are called non-catalytic receptors, as the receptors have no intrinsic tyrosine kinase activity and cannot phosphorylate their own tyrosine residues.^[2] Phosphorylation is mediated by additionally recruited kinases. A prominent member of this receptor family is the T-cell receptor.

Features and Classification

Members of the Non-catalytic tyrosine-phosphorylated receptor family share a couple of common features. The most prominent feature is the presence of conserved signalling motifs containing tyrosine residue, such as Immunoreceptor tyrosine-based activation motifs (ITAMs), in the cytoplasmic tail of the receptors. The receptor signaling pathway is initiated by ligand binding to the extracellular domains of the receptor. Upon binding, the tyrosine residues in the signaling motifs are phosphorylated by membrane-associated tyrosine kinases. The receptors themselves have no intrinsic tyrosine kinase activity. The phosphorylated NTRs, in turn, initiate a specific intracellular signaling cascades. The signaling cascade is down-regulated by dephosphorylation by protein tyrosine phosphatases. Additional characteristics of the receptor family are a rather small (< 20 nm) extracellular domain and the binding to ligands that are anchored to solid surfaces or membranes of other cells. NTRs are exclusively expressed in leukocytes.^[2]

Based on those features, about 100 distinct NTRs have been identified. The table below lists different classes of NTRs. Members of a class have a high sequence homology and typically share the same gene locus.^[2]

Family	Ligands	Examples
Antigen receptors found on T cells and B cells (T-cell receptor and B-cell receptor)	MHC class I or II loaded with peptide for T-cell receptors, soluble or surface antigens for B-cell receptor	TCR BCR
C-type lectin domain family	Glycans, Actin, MHC class I	Dectin-1, NKG2, BDCA2
CD300 family	Unknown	CD300A
Classical Fc receptor family	Fc region of antibody	FcγRI, FcγRII
Fc receptor-like family	Unknown	FCRL1
KIR family	MHC class 1	KIR2DL1, KIR3DL2, KIR2DS1
LILR family	MHC class 1	LILRB4
Natural cytotoxicity triggering receptor (NCR) family	Viral hemagglutinins, heparan sulfate proteoglycans, activation-induced C-type lectin	NKp44, NKp46, NKp30
Paired immunoglobulin-like receptor (PILR) family	PILR-associating neural protein (PANP), HSV-1 glycoprotein B	PILRA, PILRB
SIGLEC family	Endogenous and pathogen-derived sialylated glycans	SIGLEC1, SIGLEC8, SIGLEC7, SIGLEC2
CD28 family	B7 family of membrane proteins	CD28, CTLA-4, ICOS, BTLA
CD200R family	CD200	CD200R1, CD5, CD6
Signal-regulatory protein (SIRP) family	CD47, surfactant proteins e.g. SPA1	SIRPα
Signaling lymphocytic activation molecule (SLAM) family	Homophilic (bind SLAM family members), CD48	SLAMF1, SLAMF3
Collagen receptors	Collagen	LAIR1 OSCAR GPVI

Structure

NTRs are transmembrane glycoproteins with typically small ectodomains of 6 to 10 nm. NTRs have either an N-terminal or C terminal ectodomains. Ectodomains have a high sequence diversity between members.^[2] Many NTRs have an unstructured intracellular domain which contains tyrosine residues that can be phosphorylated by tyrosine kinases. Some receptors in this family, however, lack a cytoplasmic tail and therefore associate with adaptor proteins containing the same tyrosine residues.^[3] Adaptor proteins associate to their respective NTR through their transmembrane helixes carrying oppositely charged residues.^[3] The cytoplasmic domains do not contain any intrinsic tyrosine kinase activity.

Conserved tyrosine-containing motifs

Tyrosine residues of NTRs mostly appear in conserved amino acid motifs with defined sequence signatures that define whether the receptor plays an activator or inhibiting role in the cell.^[4] These motifs allow binding of proteins containing a SH2 domain.^[5] Motifs are intrinsic or in the associated adaptor subunits. Immunoreceptor tyrosine-based activation motifs (ITAMs) are short amino acid sequences that contain two tyrosine residues (Y) arranged as Yxx(L/I)x6-8Yxx(L/I), where L and I indicate Leucine or Isoleucine residue respectively (according to amino acid abbreviations), x denotes any amino acids, a subscribe 6-8 indicates a sequence of 6 to 8 amino acids in length.^[6] ITAMs recruits activating kinases to the NTR.^[5] Inhibitory signals are transduced by Immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of the signature (S/I/V/L)xYxx(I/V/L), bind to cytoplasmic tyrosine phosphatases.^[7] Immunoreceptor Tyrosine-based Switch Motifs (ITSMs) with the signature TxYxx(I/V) may induce both activator and inhibitory signals. These motifs are confined to SLAM family receptors.^[8] Finally, Immunoglobulin Tail Tyrosine Motifs (ITTMs) with a YxNM signature have been found to have a costimulatory effect.^[9]

Signalling Pathway

Biophysics of receptor-ligand binding

The signalling pathway of an NTR is induced upon binding to its respective ligand. NTRs, as they are defined, have a short ectodomain (5 - 10 nm) and bind to surface-anchored ligands. For binding to take place, the membrane of the leukocyte has to come into close proximity to the surface with the ligand. The receptor-ligand complex, once bound, spans a dimension of about 10-16 nm. Ectodomains of other surface molecules can be much larger (up to 50 nm), therefore the membrane has to bend towards the ligand, which introduces tension within the membrane. Additionally, large pulling forces can act on the complex, changing dissociation rates of the complex.^[2]

Receptor triggering

NTR triggering, the initial step of the NTR signalling pathway, involves phosphorylation of the tyrosine residues in the cytoplasmic domain of the receptor or the associated adaptor protein. Once phosphorylated, these residues recruit further signalling proteins.^[10] Phosphorylation of the tyrosine residues is performed by membrane-anchored Src family kinases (SFK) (e.g. Lck, Fyn, Lyn, Blk), while receptor protein tyrosine phosphatases (RPTP) (e.g. CD45, CD148) mediate the dephosphorylation of the same residues. SFK and RPTP are constitutively active.^[11] In an untriggered state, the activity of phosphatases dominates, keeping NTRs in an unphosphorylated state, and thus preventing signal initiation. It has been shown that inhibition of tyrosine phosphatases induces phosphorylation in NTRs and signalling even without ligand binding.^[12] It is therefore assumed that a perturbation of SFK and RPTP balance due to ligand binding, leading to stronger kinase activity and hence accumulation of phosphorylated tyrosine residues, is needed for initiation of downstream signalling.

Different mechanisms of how the balance is disturbed upon ligand binding have been suggested. The induced proximity or aggregation model suggests that upon receptor-ligand binding multiple receptors aggregate. SFKs have multiple phosphorylation sites that regulate their catalytic activity.^[13] If the kinase is associated with an NTR, aggregation brings two or more SFK into close proximity, which allows them to phosphorylate each other. Hence due to receptor aggregation, SFKs are activated leading to higher kinase activity and increased NTR phosphorylation.^[14] Evidence for this model is given by mathematical models^[14] and an experiment where artificially cross-linking NTRs led to signal induction.^[15] However, there is not sufficient evidence that receptor aggregation happens in vivo.

According to the Conformational change model, binding of a ligand induces a conformational change in the receptor such that the cytosolic domain becomes accessible for kinases. Thus phosphorylation is only possible when the receptor is bound to a ligand.^[16] However, structural studies have failed to show conformational changes.^[17]

The Kinetic segregation model proposes that RPTPs are physically excluded from NTR-ligand-binding regions. Ectodomains of RPTPs are much larger compared to NTRs and SFKs. The interaction between ligand and receptor brings the membranes into close contact, and the gap between membranes is too narrow for membrane proteins with large ectodomains to diffuse into the region. This increase the ratio of SFKs over RPTPs in the region surrounding the receptor-ligand complex. Any non-bound NTR would diffuse out of these regions too quickly to induce a downstream signal.^[18][19] Evidence for this model is given by the observation that in T cells, phosphatases CD45 and CD148 segregate from the T-cell receptor upon ligand binding.^[20] It was also shown that truncation of phosphatase ectodomains as well as the elongation of ligand ectodomains reduces the segregation and inhibits NTR triggering.^[21][22] Similar findings have been reported for Receptors,^[23] CD28 family receptors,^[24] Dectin-1.^[25]

Downstream signaling pathway

Phosphorylated tyrosine residues in cytoplasmic tails of NTRs serve as docking sites for SH2 domains of cytosolic signalling proteins. Once bound to the NTR they are activated by phosphorylation and can propagate the signal. Whether a receptor acts as an inhibitor or activator depends on the conserved tyrosine-containing motifs present in its cytoplasmic domain. Activatory motifs (ITAMs) bind kinases, such as Syk family kinases (e.g. ZAP70 for T-cell receptor) that phosphorylate a range of substrates, thereby inducing a signalling cascade leading to the activation of the leukocyte.^[26] Inhibitory motifs (ITIM) on the other hand recruit the cytoplasmic tyrosine phosphates SHP1, SHP2 and the phosphatidylinositol phosphatase SHIP-1. The phosphatases can attenuate the signal by dephosphorylating a broad range of signalling molecules.^[27]

Signal integration from multiple NTRs

At any given time, multiple NTR types can be engaged with their receptive ligands, inducing activatory, costimulatory as well as inhibitory signals. The functional response of the leukocytes depends on the integration of the signals.^[28]

References

1. Veillette A, Latour S, Davidson D (2002). "Negative regulation of immunoreceptor signaling". Annual Review of Immunology. 20: 669–707. doi:10.1146/annurev.immunol.20.081501.130710. PMID 11861615.
2. Dushek O, Goyette J, van der Merwe PA (November 2012). "Non-catalytic tyrosine-phosphorylated receptors". Immunological Reviews. 250 (1): 258–76. doi:10.1111/imr.12008. PMID 23046135. S2CID 1549902.
3. Call ME, Wucherpfennig KW (November 2007). "Common themes in the assembly and architecture of activating immune receptors". Nature Reviews. Immunology. 7 (11): 841–50. doi:10.1038/nri2186. PMID 17960150. S2CID 13500863.
4. Barrow AD, Trowsdale J (July 2006). "You say ITAM and I say ITIM, let's call the whole thing off: the ambiguity of immunoreceptor signalling". European Journal of Immunology. 36 (7): 1646–53. doi:10.1002/eji.200636195. PMID 16783855.
5. Mócsai A, Ruland J, Tybulewicz VL (June 2010). "The SYK tyrosine kinase: a crucial player in diverse biological functions". Nature Reviews. Immunology. 10 (6): 387–402. doi:10.1038/nri2765. PMC 4782221. PMID 20467426.
6. Isakov N (January 1997). "Immunoreceptor tyrosine-based activation motif (ITAM), a unique module linking antigen and Fc receptors to their signaling cascades". Journal of Leukocyte Biology. 61 (1): 6–16. doi:10.1002/jlb.61.1.6. PMID 9000531.
7. Vély F, Vivier E (September 1997). "Conservation of structural features reveals the existence of a large family of inhibitory cell surface receptors and noninhibitory/activatory counterparts". Journal of Immunology. 159 (5): 2075–7. doi:10.4049/jimmunol.159.5.2075. PMID 9278290. S2CID 40484722.
8. Veillette A, Dong Z, Pérez-Quintero LA, Zhong MC, Cruz-Munoz ME (November 2009). "Importance and mechanism of 'switch' function of SAP family adapters". Immunological Reviews. 232 (1): 229–39. doi:10.1111/j.1600-065X.2009.00824.x. PMID 19909367. S2CID 205825343.
9. Engels N, Wienands J (June 2011). "The signaling tool box for tyrosine-based costimulation of lymphocytes". Current Opinion in Immunology. 23 (3): 324–9. doi:10.1016/j.coi.2011.01.005. PMID 21324660.
10. Latour S, Veillette A (June 2001). "Proximal protein tyrosine kinases in immunoreceptor signaling". Current Opinion in Immunology. 13 (3): 299–306. doi:10.1016/S0952-7915(00)00219-3. PMID 11406361.
11. Nika K, Soldani C, Salek M, Paster W, Gray A, Etzensperger R, et al. (June 2010). "Constitutively active Lck kinase in T cells drives antigen receptor signal transduction". Immunity. 32 (6): 766–77. doi:10.1016/j.immuni.2010.05.011. PMC 2996607. PMID 20541955.
12. Chang VT, Fernandes RA, Ganzinger KA, Lee SF, Siebold C, McColl J, et al. (May 2016). "Initiation of T cell signaling by CD45 segregation at 'close contacts'". Nature Immunology. 17 (5): 574–582. doi:10.1038/ni.3392. PMC 4839504. PMID 26998761.
13. Ingley E (January 2008). "Src family kinases: regulation of their activities, levels and identification of new pathways". Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1784 (1): 56–65. doi:10.1016/j.bbapap.2007.08.012. PMID 17905674.
14. Cooper JA, Qian H (May 2008). "A mechanism for SRC kinase-dependent signaling by noncatalytic receptors". Biochemistry. 47 (21): 5681–5688. doi:10.1021/bi8003044. PMC 2614901. PMID 18444664.
15. Li HL, Davis W, Puré E (April 1999). "Suboptimal cross-linking of antigen receptor induces Syk-dependent activation of p70S6 kinase through protein kinase C and phosphoinositol 3-kinase". The Journal of Biological Chemistry. 274 (14): 9812–20. doi:10.1074/jbc.274.14.9812. PMID 10092671.
16. Xu C, Gagnon E, Call ME, Schnell JR, Schwieters CD, Carman CV, et al. (November 2008). "Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif". Cell. 135 (4): 702–13. doi:10.1016/j.cell.2008.09.044. PMC 2597348. PMID 19013279.
17. Fernandes RA, Yu C, Carmo AM, Evans EJ, van der Merwe PA, Davis SJ (September 2010). "What controls T cell receptor phosphorylation?". Cell. 142 (5): 668–9. doi:10.1016/j.cell.2010.08.018. PMID 20813251.
18. Davis SJ, van der Merwe PA (April 1996). "The structure and ligand interactions of CD2: implications for T-cell function". Immunology Today. 17 (4): 177–87. doi:10.1016/0167-5699(96)80617-7. PMID 8871350.
19. Davis SJ, van der Merwe PA (August 2006). "The kinetic-segregation model: TCR triggering and beyond". Nature Immunology. 7 (8): 803–9. doi:10.1038/ni1369. PMID 16855606. S2CID 11631728.
20. Varma R, Campi G, Yokosuka T, Saito T, Dustin ML (July 2006). "T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster". Immunity. 25 (1): 117–27. doi:10.1016/j.immuni.2006.04.010. PMC 1626533. PMID 16860761.
21. Irles C, Symons A, Michel F, Bakker TR, van der Merwe PA, Acuto O (February 2003). "CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling". Nature Immunology. 4 (2): 189–97. doi:10.1038/ni877. PMID 12496963. S2CID 31201077.
22. Choudhuri K, Wiseman D, Brown MH, Gould K, van der Merwe PA (July 2005). "T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand". Nature. 436 (7050): 578–82. Bibcode:2005Natur.436..578C. doi:10.1038/nature03843. PMID 16049493. S2CID 4319128.
23. Brzostek J, Chai JG, Gebhardt F, Busch DH, Zhao R, van der Merwe PA, Gould KG (July 2010). "Ligand dimensions are important in controlling NK-cell responses". European Journal of Immunology. 40 (7): 2050–9. doi:10.1002/eji.201040335. PMC 2909396. PMID 20432238.
24. Yokosuka T, Takamatsu M, Kobayashi-Imanishi W, Hashimoto-Tane A, Azuma M, Saito T (June 2012). "Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2". The Journal of Experimental Medicine. 209 (6): 1201–17. doi:10.1084/jem.20112741. PMC 3371732. PMID 22641383.
25. Goodridge HS, Reyes CN, Becker CA, Katsumoto TR, Ma J, Wolf AJ, et al. (April 2011). "Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse'". Nature. 472 (7344): 471–5. Bibcode:2011Natur.472..471G. doi:10.1038/nature10071. PMC 3084546. PMID 21525931.
26. Feng C, Post CB (February 2016). "Insights into the allosteric regulation of Syk association with receptor ITAM, a multi-state equilibrium". Physical Chemistry Chemical Physics. 18 (8): 5807–18. Bibcode:2016PCCP...18.5807F. doi:10.1039/c5cp05417f. PMC 4758936. PMID 26468009.
27. Coxon CH, Geer MJ, Senis YA (June 2017). "ITIM receptors: more than just inhibitors of platelet activation". Blood. 129 (26): 3407–3418. doi:10.1182/blood-2016-12-720185. PMC 5562394. PMID 28465343.
28. Ravetch JV, Lanier LL (October 2000). "Immune inhibitory receptors". Science. 290 (5489): 84–9. Bibcode:2000Sci...290...84R. doi:10.1126/science.290.5489.84. PMID 11021804.