PDZ interaction sites in integrin alpha subunits. T14853, TIP/GIPC binds to a type I recognition sequence in alpha 6A/alpha 5 and a novel sequence in alpha 6B

We used published peptide library data to identify PDZ recognition sequences in integrin alpha subunit cytoplasmic domains and found that the alpha(6)A and alpha(5) subunits contain a type I PDZ binding site (TSDA*) (asterisk indicates the stop codon). The alpha(6)A cytoplasmic domain was used for screening a two-hybrid library to find interacting proteins. The bulk of the captured cDNAs (60%) coded for TIP-2/GIPC, a cytoplasmic protein with one PDZ domain. The interaction of TIP-2/GIPC with different integrin subunits was tested in two-hybrid and in vitro binding assays. Surprisingly, TIP-2/GIPC bound strongly to the C terminus of both alpha(6)A and alpha(6)B, although the alpha(6)B sequence (ESYS*) is not suggestive of a PDZ binding site because of its polar C-terminal residue. For high affinity interaction with TIP-2/GIPC, at least one of the residues at positions -1 and -3 must be negatively charged. An aliphatic residue at position 0 increases the affinity of but is not required for this interaction. The alpha(5) integrin subunit also bound to TIP-2/GIPC. The alpha(6) integrin and TIP-2/GIPC co-localize in retraction fibers in carcinoma cells plated on laminin, a finding suggesting a functional interaction in vivo. Our results demonstrate that both splice variants of alpha(6) integrin contain a conserved PDZ binding site that enables interaction with TIP-2/GIPC. The binding site in alpha(6)B defines a new subclass of type I PDZ interaction site, characterized by a non-aliphatic residue at position 0.     

TRP1 interacting PDZ-domain protein GIPC forms oligomers and is localized to intracellular vesicles in human melanocytes

PDZ proteins coordinate assembly of protein complexes that participate in diverse biological processes. GIPC is a multifunctional PDZ protein that interacts with several soluble and membrane proteins. Unlike most PDZ proteins, GIPC contains single PDZ domain and the mechanisms by which GIPC mediates its actions remain unclear. We investigated the possibility that in lieu of multiple PDZ domains, GIPC forms multimers. Here, we demonstrate that GIPC can bind to itself and that the PDZ domain is involved in GIPC-GIPC interaction. Gel filtration, sucrose gradient centrifugation and chemical cross-linking showed that whereas bulk of cytosolic GIPC was present as monomer, oligomers with an estimated molecular mass corresponding to GIPC homotrimer were readily detectable in the membrane fraction. Modeling of GIPC PDZ domain showed feasibility of trimerization. Immunogold electron microscopy showed that GIPC is present in clusters near vesicles. Our data suggest that oligomers of GIPC mediate its functions in melanocytes.     

Interactions between GIPC-APPL and GIPC-TRP1 regulate melanosomal protein trafficking and melanogenesis in human melanocytes

By virtue of the presence of multiple protein–protein interaction and signaling domains, PDZ proteins play important roles in assembling protein complexes that participate in diverse cell biological processes. GIPC is a versatile PDZ protein that binds a variety of target proteins in different cell types. In previous studies we showed that, in epidermal melanocytes, GIPC interacts with newly synthesized melanosomal protein TRP1 in the Golgi region and proposed that this interaction may facilitate intracellular trafficking of TRP1. However, since GIPC contains a single PDZ domain and no other known protein interaction motifs, it is not known how GIPC–TRP1 interaction affects melanosome biogenesis and/or melanin pigmentation. Here, we show that in human primary melanocytes GIPC interacts with AKT-binding protein APPL (adaptor protein containing pleckstrin homology, leucine zipper and phosphotyrosine binding domains), which readily co-precipitates with newly synthesized TRP1. Knockdown of either GIPC or APPL inhibits melanogenesis by decreasing tyrosinase protein levels and enzyme activity. In melanocytes, APPL exists in a complex with GIPC and phospho-AKT. Inhibition of AKT phosphorylation using a PI3-kinase inhibitor abolishes this interaction and results in retardation TRP1 in the Golgi. These data suggest that interactions between TRP1–GIPC and GIPC–APPL–AKT provide a potential link between melanogenesis and PI3 kinase signaling.     

Domain-swapped dimerization of ZO-1 PDZ2 generates specific and regulatory connexin43-binding sites

PDZ domain scaffold proteins are capable of assembling macromolecular protein complexes in diverse cellular processes through PDZ-mediated binding to a short peptide fragment at the carboxyl tail of target proteins. How each PDZ domain specifically recognizes its target protein(s) remains a major conceptual question, as at least a few out of the several hundred PDZ domains in each eukaryotic genome share overlapping binding properties with any given target protein. Here, we show that the domain-swapped dimerization of zonula occludens-1 PDZ2 generates a distinct interface that functions together with the well-separated canonical carboxyl tail-binding pocket in each PDZ unit in binding to connexin43 (Cx43). We further demonstrate that the charge-charge interaction network formed by residues in the PDZ dimer interface and upstream residues of the Cx43 peptide not only provides the unprecedented interaction specificity for the complex but may also function as a phosphorylation-mediated regulatory switch for the dynamics of the Cx43 gap junctions. Finally, we provide evidence that such domain-swapped dimer assembly also occurs in other PDZ domain scaffold proteins. Therefore, our findings present a new paradigm for understanding how some PDZ domain proteins specifically bind to and regulate the functions of their target proteins.     

GLUT1CBP(TIP2/GIPC1) interactions with GLUT1 and myosin VI: evidence supporting an adapter function for GLUT1CBP

We identified a novel interaction between myosin VI and the GLUT1 transporter binding protein GLUT1CBP(GIPC1) and first proposed that as an adapter molecule it might function to couple vesicle-bound proteins to myosin VI movement. This study refines the model by identifying two myosin VI binding domains in the GIPC1 C terminus, assigning respective oligomerization and myosin VI binding functions to separate N- and C-terminal domains, and defining a central region in the myosin VI tail that binds GIPC1. Data further supporting the model demonstrate that 1) myosin VI and GIPC1 interactions do not require a mediating protein; 2) the myosin VI binding domain in GIPC1 is necessary for intracellular interactions of GIPC1 with myosin VI and recruitment of overexpressed myosin VI to membrane structures, but not for the association of GIPC1 with such structures; 3) GIPC1/myosin VI complexes coordinately move within cellular extensions of the cell in an actin-dependent and microtubule-independent manner; and 4) blocking either GIPC1 interactions with myosin VI or GLUT1 interactions with GIPC1 disrupts normal GLUT1 trafficking in polarized epithelial cells, leading to a reduction in the level of GLUT1 in the plasma membrane and concomitant accumulation in internal membrane structures.     

The PDZ domain of TIP-2/GIPC interacts with the C-terminus of the integrin alpha5 and alpha6 subunits.

Different cDNA libraries were screened by the yeast two-hybrid system using as a bait the cytoplasmic sequence of integrin alpha6A or alpha6B subunits. Surprisingly, the same PDZ domain-containing protein, TIP-2/GIPC, was isolated with either of the variants, although their sequences are different. Direct interaction assays with the cytoplasmic domain of the integrin alpha1--7 subunits revealed that in addition to alpha6A and alpha6B, TIP-2/GIPC reacted also with alpha5, but not other alpha integrin subunits. The specificity of the interaction was confirmed by in vitro protein binding assays with purified peptides corresponding to integrin cytoplasmic domains. Further analysis with either truncation fragments of TIP-2/GIPC or mutated integrin cytoplasmic domains indicated that the interaction occurs between the PDZ domain of TIP-2/GIPC and a consensus PDZ domain-binding sequence, SDA, present at the C-terminus of the integrin alpha5 and alpha6A subunits. The integrin alpha6B subunit terminates with a different sequence, SYS, which may represent a new PDZ domain-binding motif.     

Noncanonical Role of the PDZ4 Domain of the Adaptor Protein PDZK1 in the Regulation of the Hepatic High Density Lipoprotein Receptor Scavenger Receptor Class B,Type I (SR-BI)

The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI''s cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1''s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.     

GIPC and GAIP form a complex with TrkA: a putative link between G protein and receptor tyrosine kinase pathways

NGF initiates the majority of its neurotrophic effects by promoting the activation of the tyrosine kinase receptor TrkA. Here we describe a novel interaction between TrkA and GIPC, a PDZ domain protein. GIPC binds to the juxtamembrane region of TrkA through its PDZ domain. The PDZ domain of GIPC also interacts with GAIP, an RGS (regulators of G protein signaling) protein. GIPC and GAIP are components of a G protein-coupled signaling complex thought to be involved in vesicular trafficking. In transfected HEK 293T cells GIPC, GAIP, and TrkA form a coprecipitable protein complex. Both TrkA and GAIP bind to the PDZ domain of GIPC, but their binding sites within the PDZ domain are different. The association of endogenous GIPC with the TrkA receptor was confirmed by coimmunoprecipitation in PC12 (615) cells stably expressing TrkA. By immunofluorescence GIPC colocalizes with phosphorylated TrkA receptors in retrograde transport vesicles located in the neurites and cell bodies of differentiated PC12 (615) cells. These results suggest that GIPC, like other PDZ domain proteins, serves to cluster transmembrane receptors with signaling molecules. When GIPC is overexpressed in PC12 (615) cells, NGF-induced phosphorylation of mitogen-activated protein (MAP) kinase (Erk1/2) decreases; however, there is no effect on phosphorylation of Akt, phospholipase C-gamma1, or Shc. The association of TrkA receptors with GIPC and GAIP plus the inhibition of MAP kinase by GIPC suggests that GIPC may provide a link between TrkA and G protein signaling pathways.     

Origins of PDZ domain ligand specificity. Structure determination and mutagenesis of the Erbin PDZ domain

The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW(-4)ETW(-1)V; IC(50) = approximately 0.15 microm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp(-1) are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the beta2-beta3 loop provides a binding site for the Trp(-4) side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.     

An allosteric intramolecular PDZ-PDZ interaction modulates PTP-BL PDZ2 binding specificity

PDZ (acronym of the synapse-associated protein PSD-95/SAP90, the septate junction protein Discs-large, and the tight junction protein ZO-1) domains are abundant small globular protein interaction domains that mainly recognize the carboxyl termini of their target proteins. Detailed knowledge on PDZ domain binding specificity is a prerequisite for understanding the interaction networks they establish. We determined the binding preference of the five PDZ domains in the protein tyrosine phosphatase PTP-BL by screening a random C-terminal peptide lambda phage display library. Interestingly, the potential of PDZ2 to interact with class III-type ligands was found to be modulated by the presence of PDZ1. Structural studies revealed a direct and specific interaction of PDZ1 with a surface on PDZ2 that is opposite the peptide binding groove. Long-range allosteric effects that cause structural changes in the PDZ2 peptide binding groove thus explain the altered PDZ2 binding preference. Our results experimentally corroborate that the molecular embedding of PDZ domains is an important determinant of their ligand binding specificity.     

The binding of the PDZ tandem of syntenin to target proteins

PDZ domains are among the most abundant protein modules in the known genomes. Their main function is to provide scaffolds for membrane-associated protein complexes by binding to the cytosolic, C-terminal fragments of receptors, channels, and other integral membrane proteins. Here, using both heteronuclear NMR and single crystal X-ray diffraction, we show how peptides with different sequences, including those corresponding to the C-termini of syndecan, neurexin, and ephrin B, can simultaneously bind to both PDZ domains of the scaffolding protein syntenin. The PDZ2 domain binds these peptides in the canonical fashion, and an induced fit mechanism allows for the accommodation of a range of side chains in the P(0) and P(-)(2) positions. However, binding to the PDZ1 domain requires that the target peptide assume a noncanonical conformation. These data help explain how syntenin, and perhaps other PDZ-containing proteins, may preferentially bind to dimeric and clustered targets, and provide a mechanistic explanation for the previously reported cooperative ligand binding by syntenin's two PDZ domains.     

Chemically Modified Peptide Scaffolds Target the CFTR-Associated Ligand PDZ Domain

PDZ domains are protein-protein interaction modules that coordinate multiple signaling and trafficking pathways in the cell and that include active therapeutic targets for diseases such as cancer, cystic fibrosis, and addiction. Our previous work characterized a PDZ interaction that restricts the apical membrane half-life of the cystic fibrosis transmembrane conductance regulator (CFTR). Using iterative cycles of peptide-array and solution-binding analysis, we targeted the PDZ domain of the CFTR-Associated Ligand (CAL), and showed that an engineered peptide inhibitor rescues cell-surface expression of the most common CFTR disease mutation ΔF508. Here, we present a series of scaffolds containing chemically modifiable side chains at all non-motif positions along the CAL PDZ domain binding cleft. Concordant equilibrium dissociation constants were determined in parallel by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance techniques, confirming robust affinity for each scaffold and revealing an enthalpically driven mode of inhibitor binding. Structural studies demonstrate a conserved binding mode for each peptide, opening the possibility of combinatorial modification. Finally, we diversified one of our peptide scaffolds with halogenated substituents that yielded modest increases in binding affinity. Overall, this work validates our approach and provides a stereochemical foundation for further CAL inhibitor design and screening.     

GIPC binds to the human lutropin receptor (hLHR) through an unusual PDZ domain binding motif, and it regulates the sorting of the internalized human choriogonadotropin and the density of cell surface hLHR

By using a yeast two-hybrid screen we identified GIPC (GAIP-interacting protein C terminus), a protein with a type I PDZ domain as a novel human lutropin receptor (hLHR) binding partner. Pull-down and immunoprecipitation assays confirmed this interaction and showed that it is dependent on the PDZ domain of GIPC and the C-terminal tetrapeptide of the hLHR. To characterize the functional consequences of the GIPC-hLHR interaction, we used a small interfering RNA against GIPC to generate a clonal cell line that is deficient in GIPC. Studies with this cell line reveal that GIPC is partially responsible for the recycling of the hormone that is internalized by the hLHR and also for maintaining a relatively constant level of hLHR at the cell surface during hormone internalization.     

Distinct binding specificity of the multiple PDZ domains of INADL, a human protein with homology to INAD from Drosophila melanogaster

PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Proteins containing multiple PDZ domains often bind to different trans-membrane and intracellular proteins, playing a central role as organizers of multimeric complexes. To characterize the rules underlying the binding specificity of different PDZ domains, we have assembled a novel repertoire of random peptides that are displayed at high density at the carboxyl terminus of the capsid D protein of bacteriophage lambda. We have exploited this combinatorial library to determine the peptide binding preference of the seven PDZ domains of human INADL, a multi-PDZ protein that is homologous to the INAD protein of Drosophila melanogaster. This approach has permitted the determination of the consensus ligand for each PDZ domain and the assignment to class I, class II, and to a new specificity class, class IV, characterized by the presence of an acidic residue at the carboxyl-terminal position. Homology modeling and site-directed mutagenesis experiments confirmed the involvement of specific residues at contact positions in determining the domain binding preference. However, these experiments failed to reveal simple rules that would permit the association of the chemical characteristics of any given residue in the peptide binding pocket to the preference for specific amino acid sequences in the ligand peptide. Rather, they suggested that to infer the binding preference of any PDZ domain, it is necessary to simultaneously take into account all contact positions by using computational procedures. For this purpose we extended the SPOT algorithm, originally developed for SH3 domains, to evaluate the probability that any peptide would bind to any given PDZ domain.     

Design of a selective chemical probe for class I PDZ domains

Covalent labeling has been widely used for structural and functional analyses of proteins. To target a wide range of PDZ domains, we designed a chemical scaffold mimicking the E/D-T/S-XV peptide, which is a PDZ domain that binds ligands in higher occurrence. A chemical probe (2) that contained this moiety alkylated diverse PDZ domains, including NHERF-1 PDZ2, and differentially visualized the cellular proteome.     

GIPC participates in G protein signaling downstream of insulin-like growth factor 1 receptor.

Several recent studies have demonstrated that insulin-like growth factor (IGF)-1-induced mitogen-activated protein kinase (MAP kinase) activation is abolished by pertussis toxin, suggesting that trimeric G proteins of the G(i) class are novel cellular targets of the IGF-1 signaling pathway. We report here that the intracellular domain of the Xenopus IGF-1 receptor is capable of binding to the Xenopus homolog of mammalian GIPC, a PDZ domain-containing protein previously identified as a binding partner of G(i)-specific GAP (RGS-GAIP). Binding of xGIPC to xIGF-1 receptor is independent of the kinase activity of the receptor and appears to require the PDZ domain of xGIPC. Injection of two C-terminal truncation mutants that retained the PDZ domain blocked IGF-1-induced Xenopus MAP kinase activation and oocyte maturation. While full-length xGIPC injection did not significantly alter insulin response, it greatly enhanced human RGS-GAIP in stimulating the insulin response in frog oocytes. This represents the first demonstration that GIPC x RGS-GAIP complex acts positively in IGF-1 receptor signal transduction.     

Alteration of the C-Terminal Ligand Specificity of the Erbin PDZ Domain by Allosteric Mutational Effects

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.     

Cooperative phosphoinositide and peptide binding by PSD-95/discs large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin

PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. We screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns(4,5)P(2)-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. We identified the transmembrane protein Crumbs as a Pyd PDZ2 ligand and probed the relation between peptide and PtdIns(4,5)P(2) binding. Contrary to the prevalent view on PDZ/peptide/lipid binding, we did not find competition between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns(4,5)P(2) 6-fold. Our results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns(4,5)P(2) binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals.     

Energetic determinants of internal motif recognition by PDZ domains

PDZ domains are protein-protein interaction modules that organize intracellular signaling complexes. Most PDZ domains recognize specific peptide motifs followed by a required COOH-terminus. However, several PDZ domains have been found which recognize specific internal peptide motifs. The best characterized example is the syntrophin PDZ domain which, in addition to binding peptide ligands with the consensus sequence -E-S/T-X-V-COOH, also binds the neuronal nitric oxide synthase (nNOS) PDZ domain in a manner that does not depend on its precise COOH-terminal sequence. In the structure of the syntrophin-nNOS PDZ heterodimer complex, the two PDZ domains interact in a head-to-tail fashion, with an internal sequence from the nNOS PDZ domain binding precisely at the peptide binding groove of the syntrophin PDZ domain. To understand the energetic basis of this alternative mode of PDZ recognition, we have undertaken an extensive mutagenic and biophysical analysis of the nNOS PDZ domain and its interaction with the syntrophin PDZ domain. Our data indicate that the presentation of the nNOS internal motif within the context of a rigid beta-hairpin conformation is absolutely essential to binding; amino acids crucial to the structural integrity of the hairpin are as important or more important than residues that make direct contacts. The results reveal the general rules of PDZ recognition of diverse ligand types.