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• W2507251123 abstract "Protein-tyrosine phosphatase receptor type G (RPTPγ/PTPRG) interacts in vitro with contactin-3–6 (CNTN3–6), a group of glycophosphatidylinositol-anchored cell adhesion molecules involved in the wiring of the nervous system. In addition to PTPRG, CNTNs associate with multiple transmembrane proteins and signal inside the cell via cis-binding partners to alleviate the absence of an intracellular region. Here, we use comprehensive biochemical and structural analyses to demonstrate that PTPRG·CNTN3–6 complexes share similar binding affinities and a conserved arrangement. Furthermore, as a first step to identifying PTPRG·CNTN complexes in vivo, we found that PTPRG and CNTN3 associate in the outer segments of mouse rod photoreceptor cells. In particular, PTPRG and CNTN3 form cis-complexes at the surface of photoreceptors yet interact in trans when expressed on the surfaces of apposing cells. Further structural analyses suggest that all CNTN ectodomains adopt a bent conformation and might lie parallel to the cell surface to accommodate these cis and trans binding modes. Taken together, these studies identify a PTPRG·CNTN complex in vivo and provide novel insights into PTPRG- and CNTN-mediated signaling. Protein-tyrosine phosphatase receptor type G (RPTPγ/PTPRG) interacts in vitro with contactin-3–6 (CNTN3–6), a group of glycophosphatidylinositol-anchored cell adhesion molecules involved in the wiring of the nervous system. In addition to PTPRG, CNTNs associate with multiple transmembrane proteins and signal inside the cell via cis-binding partners to alleviate the absence of an intracellular region. Here, we use comprehensive biochemical and structural analyses to demonstrate that PTPRG·CNTN3–6 complexes share similar binding affinities and a conserved arrangement. Furthermore, as a first step to identifying PTPRG·CNTN complexes in vivo, we found that PTPRG and CNTN3 associate in the outer segments of mouse rod photoreceptor cells. In particular, PTPRG and CNTN3 form cis-complexes at the surface of photoreceptors yet interact in trans when expressed on the surfaces of apposing cells. Further structural analyses suggest that all CNTN ectodomains adopt a bent conformation and might lie parallel to the cell surface to accommodate these cis and trans binding modes. Taken together, these studies identify a PTPRG·CNTN complex in vivo and provide novel insights into PTPRG- and CNTN-mediated signaling. The complex processes that shape the nervous system include the proliferation, differentiation, and migration of neural cells, axon guidance, and the formation of synapses. At the molecular level, these intricate processes rely on interactions between cell surface receptors coupled to intracellular downstream signaling networks. Such receptors might include cadherins, Ig superfamily proteins, neurexins, neuroligins, and leucine-rich repeat proteins as well as receptor tyrosine kinases and receptor protein-tyrosine phosphatases (RPTPs) 4The abbreviations used are: RPTP, receptor protein-tyrosine phosphatase; PTPRG and PTPRZ; protein-tyrosine phosphatase receptor type G and Z, respectively; FN; fibronectin; CA, carbonic anhydrase-like; CNTN, contactin; GPI, glycophosphatidylinositol; ANOVA, analysis of variance; OS, outer segment; IS, inner segment; GCL, ganglion cell layer; PLA, proximity ligation assay; SAXS, small angle x-ray scattering; CNG, cyclic nucleotide-gated; PFA, paraformaldehyde; RMSD, root mean square deviation. (1Ensslen-Craig S.E. Brady-Kalnay S.M. Receptor protein tyrosine phosphatases regulate neural development and axon guidance.Dev. Biol. 2004; 275: 12-22Crossref PubMed Scopus (70) Google Scholar, 2Williams M.E. de Wit J. Ghosh A. Molecular mechanisms of synaptic specificity in developing neural circuits.Neuron. 2010; 68: 9-18Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Members of the RPTP family typically combine large extracellular segments and intracellular phosphatase domains, which makes them ideally suited to coordinate cell adhesion and cell signaling. Among these, the homologous protein-tyrosine phosphatase receptor type G (PTPRG) and protein-tyrosine phosphatase receptor type Z (PTPRZ) were among the first RPTPs identified in the nervous system, and their ectodomains are characterized by the presence of an inactive N-terminal carbonic anhydrase-like (CA) domain that mediates protein-protein interactions (Fig. 1A) (3Harroch S. Palmeri M. Rosenbluth J. Custer A. Okigaki M. Shrager P. Blum M. Buxbaum J.D. Schlessinger J. No obvious abnormality in mice deficient in receptor protein tyrosine phosphatase β.Mol. Cell. Biol. 2000; 20: 7706-7715Crossref PubMed Scopus (96) Google Scholar, 4Lamprianou S. Vacaresse N. Suzuki Y. Meziane H. Buxbaum J.D. Schlessinger J. Harroch S. Receptor protein tyrosine phosphatase γ is a marker for pyramidal cells and sensory neurons in the nervous system and is not necessary for normal development.Mol. Cell. Biol. 2006; 26: 5106-5119Crossref PubMed Scopus (38) Google Scholar). PTPRZ and its binding partner, the neural cell adhesion molecule contactin-1 (CNTN1), control the proliferation of oligodendrocyte precursor cells and their maturation into myelinating oligodendrocytes (5Lamprianou S. Chatzopoulou E. Thomas J.-L. Bouyain S. Harroch S. A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 17498-17503Crossref PubMed Scopus (62) Google Scholar). Less is known, however, about PTPRG, its in vivo ligands, and the physiological roles these complexes might play. Unlike PTPRZ, PTPRG is mostly expressed on neurons, although it has recently been found in some astrocytes and microglia in adult mouse brains (4Lamprianou S. Vacaresse N. Suzuki Y. Meziane H. Buxbaum J.D. Schlessinger J. Harroch S. Receptor protein tyrosine phosphatase γ is a marker for pyramidal cells and sensory neurons in the nervous system and is not necessary for normal development.Mol. Cell. Biol. 2006; 26: 5106-5119Crossref PubMed Scopus (38) Google Scholar, 6Lorenzetto E. Moratti E. Vezzalini M. Harroch S. Sorio C. Buffelli M. Distribution of different isoforms of receptor protein tyrosine phosphatase γ (Ptprg-RPTP γ) in adult mouse brain: upregulation during neuroinflammation.Brain Struct. Funct. 2014; 219: 875-890Crossref PubMed Scopus (16) Google Scholar). PTPRG interacts in vitro via its CA domain with four homologs of CNTN1 called CNTN3–6 (7Bouyain S. Watkins D.J. The protein tyrosine phosphatases PTPRZ and PTPRG bind to distinct members of the contactin family of neural recognition molecules.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 2443-2448Crossref PubMed Scopus (92) Google Scholar). All CNTNs are linked to the membrane by a glycophosphatidylinositol (GPI) anchor, suggesting that they require a co-receptor to signal across the membrane (8Shimoda Y. Watanabe K. Contactins: emerging key roles in the development and function of the nervous system.Cell Adh. Migr. 2009; 3: 64-70Crossref PubMed Scopus (112) Google Scholar, 9Zuko A. Bouyain S. van der Zwaag B. Burbach J.P.H. Contactins: structural aspects in relation to developmental functions in brain disease.Adv. Protein Chem. Struct. Biol. 2011; 84: 143-180Crossref PubMed Scopus (40) Google Scholar). CNTNs are ubiquitously expressed in the nervous system and found predominantly on neurons. Over the years, CNTN3–6 have repeatedly been associated with the sensory circuitry. For example, CNTN3–5 are expressed in neuronal layers of chick embryo retinas (10Yamagata M. Sanes J.R. Expanding the Ig superfamily code for laminar specificity in retina: expression and role of contactins.J. Neurosci. 2012; 32: 14402-14414Crossref PubMed Scopus (94) Google Scholar). Furthermore, CNTN4 is expressed by axons of specific retinal ganglion cells to target the accessory optic system (11Osterhout J.A. Stafford B.K. Nguyen P.L. Yoshihara Y. Huberman A.D. Contactin-4 mediates axon-target specificity and functional development of the accessory optic system.Neuron. 2015; 86: 985-999Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). It is also found on olfactory sensory neurons and guides their axons to specific glomeruli of the olfactory bulb, thus playing a role in odor map formation (12Kaneko-Goto T. Yoshihara S. Miyazaki H. Yoshihara Y. BIG-2 mediates olfactory axon convergence to target glomeruli.Neuron. 2008; 57: 834-846Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). CNTN5 is associated with the maturation of glutamatergic synapses of neurons of the auditory pathway (13Toyoshima M. Sakurai K. Shimazaki K. Takeda Y. Shimoda Y. Watanabe K. Deficiency of neural recognition molecule NB-2 affects the development of glutamatergic auditory pathways from the ventral cochlear nucleus to the superior olivary complex in mouse.Dev. Biol. 2009; 336: 192-200Crossref PubMed Scopus (27) Google Scholar), whereas CNTN6 participates in synapse formation between parallel fibers and Purkinje cells during cerebellar development (14Sakurai K. Toyoshima M. Takeda Y. Shimoda Y. Watanabe K. Synaptic formation in subsets of glutamatergic terminals in the mouse hippocampal formation is affected by a deficiency in the neural cell recognition molecule NB-3.Neurosci. Lett. 2010; 473: 102-106Crossref PubMed Scopus (26) Google Scholar). Less is known, however, about the physiological function of CNTN3, although cntn3 transcripts have been detected in the granule cell layers of the olfactory bulb and Purkinje cells of the cerebellum (15Yoshihara Y. Kawasaki M. Tani A. Tamada A. Nagata S. Kagamiyama H. Mori K. BIG-1: a new TAG-1/F3-related member of the immunoglobulin superfamily with neurite outgrowth-promoting activity.Neuron. 1994; 13: 415-426Abstract Full Text PDF PubMed Scopus (89) Google Scholar). In broad terms, the sites and times of CNTN3–6 expression match those of PTPRG expression in sensory neurons, such as retinal ganglion cells, the cells of the glomerulus in the olfactory bulb, and ear sensory cells (4Lamprianou S. Vacaresse N. Suzuki Y. Meziane H. Buxbaum J.D. Schlessinger J. Harroch S. Receptor protein tyrosine phosphatase γ is a marker for pyramidal cells and sensory neurons in the nervous system and is not necessary for normal development.Mol. Cell. Biol. 2006; 26: 5106-5119Crossref PubMed Scopus (38) Google Scholar, 16Horvat-Bröcker A. Reinhard J. Illes S. Paech T. Zoidl G. Harroch S. Distler C. Knyazev P. Ullrich A. Faissner A. Receptor protein tyrosine phosphatases are expressed by cycling retinal progenitor cells and involved in neuronal development of mouse retina.Neuroscience. 2008; 152: 618-645Crossref PubMed Scopus (23) Google Scholar). However, the formation of complexes between PTPRG and CNTN3–6 has yet to be confirmed in vivo. This has hampered our ability to define the potential biological roles of PTPRG·CNTN complexes. Here, our crystallographic and biochemical analyses provide molecular insights into PTPRG·CNTN3 and PTPRG·CNTN6 complexes and indicate that PTPRG·CNTN complexes share a conserved arrangement. As a first step toward defining their potential physiological roles, we investigated the association of PTPRG and CNTN3 in mouse retinas. Notably, we identified PTPRG·CNTN3 complexes in the outer segments of adult mouse photoreceptors, suggesting that PTPRG·CNTN complexes form in vivo. Furthermore, our analyses reveal that the PTPRG·CNTN3 complex can form on the surface of a single photoreceptor cell, indicating that these two proteins form a cis-complex. This finding led us to consider the possibility that CNTNs might interact with PTPRG when expressed either on the same cell or on apposing cells. We thus wondered whether specific structural features in the extracellular regions of CNTNs could facilitate such interactions. Our crystallographic analyses indicate that the FN regions in CNTNs adopt a bent conformation that might place the ectodomains of CNTNs parallel to the cell surface, where they would bind to PTPRG in either cis or trans configurations. In a broader context, the sum of our analyses raises the possibility that PTPRG is a versatile signaling partner for CNTN3–6, possibly functioning as co-receptor when expressed on the same cell membrane and as a ligand when expressed on a distinct cell membrane. The results of previous affinity isolation assays using CNTN-transfected cells and a PTPRG resin suggested that the CA domain of PTPRG interacts with CNTN3–6, whereas the CA domain of the homologous PTPRZ interacts with CNTN1 only (7Bouyain S. Watkins D.J. The protein tyrosine phosphatases PTPRZ and PTPRG bind to distinct members of the contactin family of neural recognition molecules.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 2443-2448Crossref PubMed Scopus (92) Google Scholar), yet it was important to test whether these interactions could occur on the surface of cells. We thus expressed the CA and FN domains of PTPRG as a fusion protein with human IgG Fc and applied it to HEK293 cells transfected with full-length CNTNs (Fig. 1, A and B). In these experiments, PTPRG-Fc fusion proteins bound to cells transfected with CNTN3–6 but not to cells transfected with CNTN1 or CNTN2, thus demonstrating that the ectodomain of PTPRG interacts specifically with CNTN3–6 expressed at the cell surface. An earlier crystal structure of PTPRG(CA) bound to domains Ig1–Ig4 of mouse CNTN4 made it possible to identify CNTN4 residues that mediate interactions with PTPRG (7Bouyain S. Watkins D.J. The protein tyrosine phosphatases PTPRZ and PTPRG bind to distinct members of the contactin family of neural recognition molecules.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 2443-2448Crossref PubMed Scopus (92) Google Scholar). Sequence analyses of CNTN3–6 indicate that these residues are strictly conserved, indicating that PTPRG·CNTN complexes may be arranged similarly. However, it remains unclear whether there might be quantitative differences among the interactions between PTPRG and full-length CNTN family members. To address this question, we designed a protein-protein binding assay utilizing AlphaScreen technology in which a luminescent signal is emitted when a biotinylated form of PTPRG(CA) attached to a donor bead associates with a full-length CNTN expressed as an Fc fusion protein bound to an acceptor bead (supplemental Fig. S1A) (17Tolbert W.D. Daugherty J. Gao C. Xie Q. Miranti C. Gherardi E. Vande Woude G. Xu H.E. A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 14592-14597Crossref PubMed Scopus (45) Google Scholar). The binding strength is measured indirectly in a competitive-binding assay format whereby a soluble form of PTPRG(CA) inhibits the interactions between the proteins immobilized on the beads (Fig. 1C and supplemental Table S1). The assays are thus conducted with a truncated PTPRG and dimerized CNTNs so that the IC50 values obtained are appropriate for comparing binding between PTPRG and CNTNs but might not, however, reflect the true binding constants between PTPRG and CNTNs expressed at the cell surface. Under these conditions, the IC50 values for PTPRG/CNTN interactions range from 235 nm (PTPRG/CNTN4) to 519 nm (PTPRG/CNTN6) and are similar to the IC50 value of 332 nm obtained for PTPRZ/CNTN1 (supplemental Fig. S1B and Table S1). One-way ANOVA statistics indicated that only IC50 differences measured for CNTN4 versus CNTN3 or CNTN6 and for CNTN5 versus CNTN6 were statistically significant (Fig. 1D). However, it remains unclear whether these variations in binding affinities are significant enough to suggest differences in the physiological roles of PTPRG·CNTN complexes. Previous work had demonstrated that the minimal PTPRG-binding site on CNTN3–6 comprises domains Ig2-Ig3 (7Bouyain S. Watkins D.J. The protein tyrosine phosphatases PTPRZ and PTPRG bind to distinct members of the contactin family of neural recognition molecules.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 2443-2448Crossref PubMed Scopus (92) Google Scholar). Given the high level of sequence identity among CNTN3–6, we wondered whether there could remain differences in the details of the interactions between PTPRG and CNTN3–6. We addressed this by determining the co-crystal structures of PTPRG(CA) bound to the Ig2-Ig3 segments of CNTN3 and CNTN6 (Table 1 and Fig. 2, A and B). Overall, the crystal structures of the PTPRG·CNTN3 and PTPRG·CNTN6 complexes are similar to the structures of the PTPRG·CNTN4 and PTPRZ·CNTN1 complexes (5Lamprianou S. Chatzopoulou E. Thomas J.-L. Bouyain S. Harroch S. A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 17498-17503Crossref PubMed Scopus (62) Google Scholar, 7Bouyain S. Watkins D.J. The protein tyrosine phosphatases PTPRZ and PTPRG bind to distinct members of the contactin family of neural recognition molecules.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 2443-2448Crossref PubMed Scopus (92) Google Scholar) (Fig. 2C). The binding site includes a β-hairpin loop (residues 288–301) that contacts both Ig2 and Ig3 domains and a short stretch (residues 225–229) that interacts only with Ig3 (Fig. 2). The interface areas and shape complementarity coefficients are 1,668 Å2/0.62 for the PTPRG·CNTN3 complex and 1,446 Å2/0.68 for the PTPRG·CNTN6 complex. These values are comparable with the published values for the PTPRG·CNTN4 and PTPRZ·CNTN1 complexes. Overall, these analyses indicate that the complexes of PTPRG and PTPRZ bound to their cognate CNTN partners share a conserved arrangement with one another.TABLE 1Data collection and refinement statisticsCNTN3(Ig2-Ig3)·PTPRG(CA)CNTN6(Ig2-Ig3)·PTPRG(CA)CNTN5(Ig1–Ig4)CNTN1 (FN1–FN3)SeCNTN2 (FN1–FN3)CNTN2 (FN1–FN3)CNTN3 (FN1-FN3)CNTN3 (Ig5-FN2)CNTN4 (FN1–FN3)CNTN5 (FN1–FN3)CNTN6 (FN1–FN3)Data collectionBeamlineAPS 22-IDAPS 22-BMAPS 22-BMAPS 22-IDAPS 22-BMAPS 22-IDAPS 22-IDAPS 22-BMAPS 22-BMAPS 22-IDAPS 22-IDWavelength (Å)1.01.01.01.00.979161.01.01.00.979331.01.0Unique reflections31,16670,48214,07048,70149,12128,3578,97620,57813,38714,28521,656Resolution (Å)50–2.650–2.050–2.650–2.550–1.830–2.050–2.850–2.450–2.550–2.750–2.7Space groupP21212P212121C2P21P21212P21212C2P212121C2221C2221P212121Unit cella, b, c (Å)74.14, 90.53, 147.4578.64, 113.53, 117.05179.98, 50.66, 51.0287.48, 49.87, 163.30124.82, 40.85, 83.11124.39, 40.67, 82.60185.10, 39.03, 52.4058.22, 76.93, 115.8294.79, 144.3, 55.4083.77, 154.52, 90.4286.74, 90.85, 99.35α, β, γ (degrees)90.0, 90.0, 90.090.0, 90.0, 90.090.0, 101.67, 90.090.0, 97.12, 90.090.0, 90.0, 90.090.0, 90.0, 90.090.0, 96.9, 90.090.0, 90.0, 90.090.0, 90.0, 90.090.0, 90.0, 90.090.0, 90.0, 90.0RsymaRsym = Σh Σi|Ii(h) − 〈I(h)〉|/Σh Σi Ii(h), where Ii(h) is the ith measurement of reflection h and 〈I(h)〉 is a weighted mean of all measurements of h.0.134 (0.588)bValues in parentheses apply to the high resolution shell.0.096 (0.593)0.154 (0.435)0.075 (0.380)0.082 (0.479)0.110 (0.508)0.139 (0.377)0.165 (0.548)0.08 (0.460)0.112 (0.418)0.155 (0.515)CompletenessbValues in parentheses apply to the high resolution shell. (%)99.6 (96.3)98.7 (89.2)98.8 (90.7)99.2 (92.3)99.1 (92.7)97.8 (89.3)97.7 (90.0)98.2 (87.5)98.3 (87.4)85.7 (56.3)97.9 (92.5)Redundancy11.4 (5.3)7.1 (5.3)6.5 (3.5)6.9 (4.7)7.0 (5.6)5.9 (3.2)8.9 (5.8)11.9 (7.9)6.1 (4.2)12 (5.9)7.4 (4.8)I/iΣi7.2 (2.3)19.5 (2.0)11.4 (2.3)20.4 (3.3)19.8 (2.7)12.5 (1.6)15.0 (4)14.8 (2.4)20.7 (2.3)17.35 (3.6)8.4 (2.1)RefinementMolecules in asymmetric unit2 × 22 × 214111112Resolution (Å)49.2–2.624.9–2.037.0–2.643.5–2.529.1–2.047.8–2.843.1–2.428.7–2.538.6–2.743.4–2.7Rworkcr = Σh Fo(h) − Fc(h)|/Σh|Fo|. Rwork and Rfree were calculated from the working and test reflection sets, respectively./Rfree0.187/0.2490.167/0.2170.195/0.2640.204/0.2470.190/0.2260.183/0.2440.203/0.2530.190/0.2550.194/0.2310.200/241No. of atoms7,2918,0393,0869,5722,4912,1353,1902,3942,2824,715Protein7,2057,3922,9689,2672,3012,1103,0322,3252,2584,672Ligand27112287565Water595359023019025152691943Root mean square deviationsIdeal bonds (Å)0.0090.0070.0030.0040.0070.010.0090.0090.0090.004Ideal angles (degrees)1.101.050.830.841.061.211.281.121.130.92Average B factors (Å2)71.240.950.163.739.25741.261.693.742.8Protein71.340.449.663.939.157.241.361.893.842.9Ligand98.371.988.085.562.9121.1Water49.941.651.650.240.538.936.953.569.734.9Ramachandran statisticsFavored (%)94979596989596979497Allowed (%)6354254363PDB accession code5E5R5E5U5E4I5E535E7L5E4Q5I995E4S5E525E55a Rsym = Σh Σi|Ii(h) − 〈I(h)〉|/Σh Σi Ii(h), where Ii(h) is the ith measurement of reflection h and 〈I(h)〉 is a weighted mean of all measurements of h.b Values in parentheses apply to the high resolution shell.c r = Σh Fo(h) − Fc(h)|/Σh|Fo|. Rwork and Rfree were calculated from the working and test reflection sets, respectively. Open table in a new tab Broadly, interfaces for the PTPRG·CNTN complexes can be divided into four parts (Fig. 3, A–D): 1) a predominantly hydrophobic site that comprises residues on the base of the PTPRG(CA) β-hairpin loop; 2) a 5-amino acid stretch interacting with corresponding residues in the Ig3 domain of CNTN3/6; 3) an antiparallel β-sheet formed by the β-hairpin loop and an antiparallel three-strand β-sheet in domain Ig2 of CNTN3/6; and 4) the tip of the β-hairpin loop formed by residues Gln293–Val296 that interacts with domain Ig2 of CNTN3/6. In the PTPRG·CNTN3/6 complexes, site 1 is formed by PTPRG residues Phe288, Thr289, Thr290, and Tyr301 and CNTN3/6 residues Met222 and Tyr225. Site 2 includes residues Val225-Lys229 in PTPRG and residues Glu226, Pro227, Lys228, and Asn306 in CNTN3/6. In site 3, the combined 5-strand antiparallel β-sheet is stabilized by hydrogen bonds between the main chain atoms of His295–Glu300 in PTPRG and Gly139–Leu143 in CNTN3/6. In addition, the side chain of CNTN3/6 Gln138 side chain forms two hydrogen bonds with the main chain atoms of Val299 and side chain atoms of Glu300 in PTPRG. Finally, in site 4, the tip of the PTPRG(CA) β-hairpin loop rests against the Ig2 domain of CNTN3/6, and the side chain of His295 packs against Arg129 in particular. Although the contacts found at sites 1–3 are conserved in all PTPRG·CNTN structures, the side chains of residues Gln293, Asp294, His295, and Val296 in site 4 adopt distinct conformations (Fig. 3D), which suggests that this region is flexible and might only play a minor role in mediating protein-protein interactions. The conserved contacts at the interfaces of complexes formed by PTPRG with CNTN3, -4, and -6 (Fig. 3E) are consistent with the comparable IC50 values determined in our binding assays (Fig. 1, C and D). Finally, it was not possible to obtain co-crystals of PTPRG and CNTN5, but comparison of the PTPRG-binding site in CNTN4 and the corresponding region in CNTN5 shows that they are essentially identical (Fig. 3F). This finding, along with the similar affinities between PTPRG(CA) and CNTN3–6, strongly suggests that the binding mode observed for PTPRG(CA) and CNTN3, -4, and -6 is conserved for PTPRG and CNTN5. Although most of the interactions between PTPRG and its cognate CNTN partners are mediated by the β-hairpin loop in the CA domain of PTPRG (Fig. 3), comparison of the complexes PTPRG forms with CNTN3, -4, and -6 indicates that the tip of the loop adopts distinct conformations, arguing that it might not mediate essential contacts (Fig. 3E). We designed three mutant forms of the CA domain of mouse PTPRG to define the contribution of each region to the complex formation (Fig. 4A): 1) a deletion mutant in which sites 3 and 4 are eliminated as residues 290–299 of the β-hairpin loop are replaced by the tripeptide ASA; 2) a form including the mutations H295A/V296A at the tip of the β-hairpin in site 4; and 3) a form that includes two alanine mutations in the short loop in site 2 (H226A/K229A). All proteins behaved comparably with wild-type PTPRG(CA) and in particular were monomeric as determined by size exclusion chromatography. Furthermore, the structural integrity of the two alanine site-directed mutants was verified by circular dichroism spectropolarimetry, which indicates that the mutations did not alter the secondary structures of these mutated domains (supplemental Fig. S2). Because our analyses strongly suggest that all PTPRG·CNTN complexes share a similar binding mode, the binding activities of these mutant proteins were only tested for CNTN4 (Fig. 4B). The β-hairpin deletion mutant did not inhibit the interaction between PTPRG(CA) and an IgG Fc fusion of mouse CNTN4, which mirrors the effect of the β-hairpin deletion on the binding of PTPRZ and CNTN1 (5Lamprianou S. Chatzopoulou E. Thomas J.-L. Bouyain S. Harroch S. A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 17498-17503Crossref PubMed Scopus (62) Google Scholar). In contrast, the mutations H295A/V296A only increased the IC50 by ∼4-fold, consistent with our observation that contacts in site 4 might not be essential for complex formation. The decrease in binding may result from the loss of the hydrophobic Val296 side chain, which interacts with CNTN Leu142 and Val132 in CNTN3, -4, and -6. Removing the His295 side chain prevents van der Waals contacts with the side chain atoms of Arg129 and Cys144. However, one expects that the substitution to alanine residues would preserve the main chain interactions, including formation of the five-strand antiparallel β-sheet (Fig. 2C). Finally, mutating His226 and Lys229 to alanine abolished the CNTN4 binding activity (Fig. 4B). Indeed, His226 mediates conserved non-polar interaction with Tyr225, Pro227, and Lys228 and a hydrogen bond with the main-chain oxygen atom of Glu298. Substituting Lys229 to alanine disrupts the Lys229-Glu226 salt bridge and the Lys229-Asn306 hydrogen bond (Fig. 3, B and D). Overall, these results confirm the importance of the β-hairpin loop in complex formation but also indicate that contacts located in site 2 mediate essential interactions at the interface. Despite strong lines of evidence that PTPRG interacts specifically with CNTN3–6 in vitro, the identification of such complexes in vivo has yet to be investigated thoroughly. This question was addressed in mouse retinas, where PTPRG and CNTN3–5 are expressed (10Yamagata M. Sanes J.R. Expanding the Ig superfamily code for laminar specificity in retina: expression and role of contactins.J. Neurosci. 2012; 32: 14402-14414Crossref PubMed Scopus (94) Google Scholar, 16Horvat-Bröcker A. Reinhard J. Illes S. Paech T. Zoidl G. Harroch S. Distler C. Knyazev P. Ullrich A. Faissner A. Receptor protein tyrosine phosphatases are expressed by cycling retinal progenitor cells and involved in neuronal development of mouse retina.Neuroscience. 2008; 152: 618-645Crossref PubMed Scopus (23) Google Scholar). In simple terms, the retina includes three major neuronal layers (Fig. 5A). Light is first detected by photopigments in the outer segments of rod and cone photoreceptor cells spanning the outer segment (OS), inner segment (IS), and outer nuclear layer. Information is then transmitted to the bipolar cells found in the inner nuclear layer and then to ganglion cells in the ganglion cell layer (GCL) before being sent to the visual cortex by the optic nerve. The outer plexiform layer and inner plexiform layer include synapses between the photoreceptors and bipolar cells and between the bipolar cells and ganglion cells, respectively. As a first step, we analyzed the distribution of PTPRG and CNTN3 by immunohistochemistry using antibodies from commercial sources that we validated in our laboratory (Fig. 5B and supplemental Fig. S3, A and B). Consistent with previous findings (16Horvat-Bröcker A. Reinhard J. Illes S. Paech T. Zoidl G. Harroch S. Distler C. Knyazev P. Ullrich A. Faissner A. Receptor protein tyrosine phosphatases are expressed by cycling retinal progenitor cells and involved in neuronal development of mouse retina.Neuroscience. 2008; 152: 618-645Crossref PubMed Scopus (23) Google Scholar), a strong signal could be observed for PTPRG in the OSs, ISs, inner plexiform layer, and GCL. By contrast, CNTN3 was mostly localized to the OS, where it overlapped with PTPRG (Fig. 5B). We decided to assess whether PTPRG and CNTN3 form complexes in the OS by using an in situ proximity ligation assay (PLA) (18Söderberg O. Gullberg M. Jarvius M. Ridderstråle K. Leuchowius K.-J. Jarvius J. Wester K. Hydbring P. Bahram F. Larsson L.-G. Landegren U. Direct observation of individual endogenous protein complexes in situ by proximity ligation.Nat. Methods. 2006; 3: 995-1000Crossref PubMed Scopus (1754) Google Scholar, 19Hayashi M. Majumdar A. Li X. Adler J. Sun Z. Vertuani S. Hellberg C. Mellberg S. Koch S. Dimberg A. Koh G.Y. Dejana E. Belting H.-G. Affolter M. Thurston G. et al.VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation.Nat. Commun. 2013; 4: 1672Crossref PubMed Scopus (100) Google Scholar). In these experiments, we detected the interaction of endogenous PTPRG and CNTN3 in vivo by using antibodies to determine whether these proteins are in close proximity with one another (<40 nm). Consistent with our immunohistochemistry experiments, a strong signal was observed in the OS of the photoreceptors, suggesting that PTPRG and CNTN3 form a complex in photoreceptors (Fig. 5C). In contrast, no signal was observed when the primary antibody against CNTN3 was omitted (Fig. 5D). Furthermore, we observed PLA spots in the OS when we conducted assays using a distinct PTPRG antibody raised against its CA domain, which confirms the specificity of the interactions identified between CNTN3 and PTPRG (supplemental Fig. S4). Because our immunohist" @default.
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• W2507251123 title "Structural Basis for Interactions Between Contactin Family Members and Protein-tyrosine Phosphatase Receptor Type G in Neural Tissues" @default.
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