Structural Basis for Interactions Between Contactin Family Members and Protein-tyrosine Phosphatase Receptor Type G in Neural Tissues

Roman M Nikolaienko,Michal Hammel,Véronique Dubreuil,Rana Zalmai,David R Hall,Nurjahan Mehzabeen,Sebastian J Karuppan,Sheila Harroch,Salvatore L Stella,Samuel Bouyain

doi:10.1074/jbc.m116.742163

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.

Highlights

Protein-tyrosine phosphatase receptor type G (RPTP␥/ 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
PTPRG Interacts with CNTN3– 6 —The results of previous affinity isolation assays using CNTN-transfected cells and a PTPRG resin suggested that the carbonic anhydrase-like (CA) domain of PTPRG interacts with CNTN3– 6, whereas the CA domain of the homologous phosphatase receptor type Z (PTPRZ) interacts with CNTN1 only [7], yet it was important to test whether these interactions could occur on the surface of cells
PTPRG-Fc fusion proteins bound to cells transfected with CNTN3– 6 but not to cells transfected with CNTN1 or CNTN2, demonstrating that the ectodomain of PTPRG interacts with CNTN3– 6 expressed at the cell surface

Summary

Results

PTPRG Interacts with CNTN3– 6 —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 [7], yet it was important to test whether these interactions could occur on the surface of cells. Sequence analyses of CNTN3– 6 indicate that these residues are strictly conserved, indicating that PTPRG1⁄7CNTN complexes may be arranged It remains unclear whether there might be quantitative differences among the interactions between PTPRG and full-length CNTN family members. 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.

Data collection and refinement statistics

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Discussion