Syntenin-Syndecan Binding Requires Syndecan-Synteny and the Co-operation of Both PDZ Domains of Syntenin

Johan J Grootjans,Gunter Reekmans,Helga Ceulemans,Guido David

doi:10.1074/jbc.m002459200

Johan J Grootjans, Gunter Reekmans + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m002459200

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Abstract

Syntenin is an adaptor-like molecule that binds to the cytoplasmic domains of all four vertebrate syndecans. Syntenin-syndecan binding involves the C-terminal part of syntenin that contains a tandem of PDZ domains. Here we provide evidence that each PDZ domain of syntenin can interact with a syndecan. Isolated or combined mutations of the carboxylate binding lysines in the inter-betaAbetaB loops and of the alphaB1 residues in either one or both the PDZ domains of syntenin all reduce syntenin-syndecan binding in yeast two-hybrid, blot-overlay, and surface plasmon resonance assays. PDZ2 mutations have more pronounced effects on binding than PDZ1 mutations, but complete abrogation of syntenin-syndecan binding requires the combination of both the lysine and the alphaB1 mutations in both the PDZ domains of syntenin. Isothermal calorimetric titration of syntenin with syndecan peptide reveals the presence of two binding sites in syntenin. Yet, unlike a tandem of two PDZ2 domains and a reconstituted PDZ1+PDZ2 tandem, a tandem of two PDZ1 domains and isolated PDZ1 or PDZ2 domains do not interact with syndecan bait. We conclude to a co-operative binding mode whereby neither of these two PDZ domains is sufficient by itself but where PDZ2 functions as a "major" or "high affinity" syndecan binding domain, and PDZ1 functions as an "accessory" or "low affinity" syndecan binding domain. The paired, but not the isolated PDZ domains of syntenin bind also strongly to the immobilized cytoplasmic domains of neurexin and B-class ephrins. By inference, these data suggest a model whereby recruitment of syntenin to membrane surfaces requires two compatible types of bait that are in "synteny" (occurring together in location) and engages both PDZ domains of syntenin. The synteny of compatible bait may result from the assemblies and co-assemblies of syndecans and other similarly suited partners in larger supramolecular complexes. In general, an intramolecular combination of PDZ domains that are weak, taken individually, would appear to be designed to detect rather than drive the formation of specific molecular assemblies.

Highlights

Syndecans are type-I membrane proteins that are substituted with heparan sulfate
The cytoplasmic domains of the syndecans were shown to interact with syntenin [2] and CASK [3, 4], two proteins that belong to the larger family of proteins that contain one or several PDZ domains (PDZ proteins)
CASK, originally identified as a membrane-associated guanylate kinase homolog that binds to the t-YYV sequence of neurexin [13] and recently shown to bind to syndecans [3, 4], is one of these proteins with single PDZ domains that work in isolation

Summary

EXPERIMENTAL PROCEDURES

CDNA Constructs—Mutant syndecan and syntenin cDNAs were generated with the QuickChange site-directed mutagenesis kit (Stratagene). cDNAs encoding syntenin-PDZ1 (amino acid Gly-102 to Pro194) or PDZ2 (amino acid Phe-195 to Pro-271) were constructed by PCR with the indicated primer pairs (5Ј-GGGATCCTGTTTGAACGGACGATTACCATG and 5Ј-TCAGATCTTAGGCATGATTGTAATAGTAAC for PDZ1; 5Ј-GGGATCCGTAGAGCAGAAATTAAGCAAG and 5Ј-TCAGATCTTGGGCGTGTCACGAATGG for PDZ2) and ligated to one another (using the BamHI and BglII restriction sites) to encode tandems of PDZ1-PDZ1, PDZ1-PDZ2, or PDZ2-PDZ2 (verified by restriction mapping and DNA sequencing). cDNA encoding the cytoplasmic domain of neurexin was constructed by PCR using the primers 5Ј-AGGATCCCCATGTATAAGTACCGCAATCG and 5Ј-TGCGAATTCCGTAAGAGAAGCTCTTGAGGC. B-ephrin cDNAs were cloned into the pGEX-5x-2 expression vector (Amersham Pharmacia Biotech) to create fusion proteins between GST and the cytoplasmic domains of these membrane proteins. Vectors encoding fusion proteins between GST-myc and wild-type syntenin or paired syntenin-PDZ domains were constructed by inserting the corresponding cDNAs in the BamHI site of this modified vector. Blotting Assays—Purified GST fusion proteins or isopropyl ␤-D-thiogalactoside-induced BL21/ER2566 cells expressing these fusion proteins were re-suspended in SDS sample buffer and boiled for 5 min. CDNA-encoding syntenin was produced by PCR using the primers 5Ј-GGAATTCCATATGTCTCTCTATCCATCTCTCGAAG and 5Ј-GGCCGCTCTTCCGCAAGCCTCAGGAATGGTGTGGTCCA and cloned in the NdeI and Sap I restriction sites of the pTYB1 vector to produce an in-frame fusion between the C terminus of syntenin and the N terminus of an intein-chitin binding domain fusion protein. The obtained titration thermogram was analyzed with MicroCalTMOriginTM 5.0 software

RESULTS

Findings

DISCUSSION