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

Abstract

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. 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 PTPRG⅐CNTN3-6 complexes share similar binding affinities and a conserved arrangement. Furthermore, as a first step to identifying PTPRG⅐CNTN 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 PTPRG⅐CNTN 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) 4 (1, 2). 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 proteinprotein interactions (Fig. 1A) (3, 4). 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 (5). 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 (4, 6). PTPRG interacts in vitro via its CA domain with four homologs of CNTN1 called CNTN3-6 (7). All CNTNs are linked to the membrane by a glycophosphatidylinositol (GPI) anchor, suggesting that they require a co-receptor to signal across the membrane (8, 9). CNTNs a...

3D Images of Neuronal Adhesion Molecule Contactin-2 Reveal an Unanticipated Two-State Architecture

“…3E-G). To understand the structure of entire extracellular domain, the domains of lg5 -lg6 and FNIII1 -FNIII2 were docked by the homology model of CNTN3 Ig5 -FNIII_2 fragment (PDBID: 5I99; 45.3% identity to the corresponding domains in human CNTN2), while the domain FNIII3 -FNIII4 were docked by the homology model of mouse CNTN2 FNIII_1 -FNIII_3 (PDBID: 5E7L) in each IPET 3D density maps 15 . Similar docking studies in the IPET maps of Category 2 particles revealed a fundamentally different arrangement with the first four Ig domains in an extended conformation resembling beads-on-a-string ( Fig.…”

“…To date, all of the crystal structures of the Ig1-Ig4 headpiece in CNTNs and related proteins have revealed a compact horseshoe-shaped configuration [12][13][14][15][16][17][18][19][20][21] . However, the data presented here reveal that most of the CNTN2 particles in our EM images in fact do not adopt a closed horseshoe-shaped Ig1-Ig4 headpiece.…”

“…All CNTNs consist of six Ig-like domains connected to four fibronectin III-like domains; the ectodomain is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor 11 . Crystal structures have revealed that the first four Ig domains of CNTN2 form a compact moiety arranged in a 'horseshoe'-shaped structure 12,13 , a configuration that is also seen in the Ig1-Ig4 fragment of CNTN3, CNTN4, CNTN5 and CNTN6 14,15 . To form the U-shaped arrangement, Ig1 reaches across to interact with Ig4, while Ig2 binds to Ig3 and is connected also via a six residue linker.…”

“…This horseshoe-shaped configuration is also observed in other proteins containing a concatenation of 4 or more Ig domains, such as the cell adhesion molecules neurofascin, DSCAM, DCC, and the sidekicks, as well as in hemolin, a protein involved in insect immunity [16][17][18][19][20][21] . Crystallographic studies on other fragments have revealed that CNTN3 Ig5-FNIII_2 adopts an extended conformation while FNIII_1-FNIII_3 in CNTN1 through CNTN6 adopts an L-shaped conformation with a kink between FNIII_2 and FNIII_3 15 .…”

“…The CNTN Ig1-Ig4 headpiece is important functionally because it binds to protein partners. Ig1-Ig4 of CNTN2 binds NgCAM (a six Ig and five FNIII domain containing neural cell surface adhesion molecule) 22 , while the Ig1-Ig4 regions of CNTN3 through CNTN6 (but not CNTN1 or CNTN2) bind the carbonic anhydrase domain of PTPRG 14,15 and that of DCC binds to draxin 23 . The Ig1-Ig4 headpiece is also thought to be important for homophilic binding between CNTN2 molecules because mutations in this region are disruptive in cell aggregation and binding studies 12,22 .…”