A transmembrane leucine zipper is required for activation of the dimeric receptor tyrosine kinase DDR1

Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for self-association in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper.     

Antagonism of LIN-17/Frizzled and LIN-18/Ryk in nematode vulva induction reveals evolutionary alterations in core developmental pathways

Most diversity in animals and plants results from the modification of already existing structures. Many organ systems, for example, are permanently modified during evolution to create developmental and morphological diversity, but little is known about the evolution of the underlying developmental mechanisms. The theory of developmental systems drift proposes that the development of conserved morphological structures can involve large-scale modifications in their regulatory mechanisms. We test this hypothesis by comparing vulva induction in two genetically tractable nematodes, Caenorhabditis elegans and Pristionchus pacificus. Previous work indicated that the vulva is induced by epidermal growth factor (EGF)/RAS and WNT signaling in Caenorhabditis and Pristionchus, respectively. Here, we show that the evolution of vulva induction involves major molecular alterations and that this shift of signaling pathways involves a novel wiring of WNT signaling and the acquisition of novel domains in otherwise conserved receptors in Pristionchus vulva induction. First, Ppa-LIN-17/Frizzled acts as an antagonist of WNT signaling and suppresses the ligand Ppa-EGL-20 by ligand sequestration. Second, Ppa-LIN-18/Ryk transmits WNT signaling and requires inhibitory SH3 domain binding motifs, unknown from Cel-LIN-18/Ryk. Third, Ppa-LIN-18/Ryk signaling involves Axin and β-catenin and Ppa-axl-1/Axin is epistatic to Ppa-lin-18/Ryk. These results confirm developmental system drift as an important theory for the evolution of organ systems and they highlight the significance of protein modularity in signal transduction and the dynamics of signaling networks.     

Plexin B regulates Rho through the guanine nucleotide exchange factors leukemia-associated Rho GEF (LARG) and PDZ-RhoGEF

Plexins represent a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Emerging evidence implicates Rho GTPases in these biological events. However, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to Rho is still unknown. Here we show that Plexin B2 associates directly with two members of a recently identified family of Dbl homology/pleckstrin homology containing guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and Leukemia-associated Rho GEF (LARG). This physical interaction is mediated by their PDZ domains and a PDZ-binding motif found only in Plexins of the B family. In addition, we show that ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently and to induce the reorganization of the cytoskeleton. Moreover, overexpression of the PDZ domain of PDZ-RhoGEF but not its regulator of G protein signaling domain prevents cell rounding and neurite retraction of differentiated PC12 cells induced by activation of endogenous Plexin B1 by semaphorin 4D. The association of Plexins with LARG and PDZ-RhoGEF thus provides a direct molecular mechanism by which semaphorins acting on Plexin B can control Rho, thereby regulating the actin-cytoskeleton during axonal guidance and cell migration.     

Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels

The three connexins expressed in the ocular lens each contain PDZ domain-binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell-cell communication in HeLa cells, whereas the addition of a seven-amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain-binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.     

A sequence motif in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization

A mechanism by which ligand binding to the extracellular domain of a growth factor receptor causes activation of its cytoplasmic tyrosine kinase domain is that binding promotes receptor dimerization. Recently we proposed a model in which dimerization of the transmembrane alpha-helices in one member of this family, rat neu, is mediated by the presence of three specific residues. This paper shows that a similar sequence motif is observed in 18 of the 20 transmembrane alpha-helices of the tyrosine kinase family of growth factor receptors. The motif encompasses a five residue segment in which position 0 (P0) requires a small side chain (Gly, Ala, Ser, Thr or Pro), P3 an aliphatic side chain (Ala, Val, Leu or Ile) and P4 only the smallest side chains (Gly or Ala). In addition other features of the transmembrane sequences are reported. It is concluded that the dimerization of transmembrane alpha-helices may be a general mechanism of tyrosine kinase activation in this family of growth factor receptors.     

A cytoplasmic domain mutation in ClC-Kb affects long-distance communication across the membrane

Background

ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter''s Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter''s Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix.

Methodology/Principal Findings

Two-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface.

Conclusions

The R538P mutation in ClC-Kb, which leads to Bartter''s Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change – ranging from the cytoplasmic side of the protein to the extracellular side of the protein – is involved in the Ca²⁺-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel''s electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca²⁺. This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure.     

The XcpR protein of Pseudomonas aeruginosa dimerizes via its N-terminus

Extracellular protein secretion by the main terminal branch of the general secretory pathway in Pseudomonas aeruginosa requires a secretion machinery comprising the products of at least 12 genes. One of the components of this machinery, the XcpR protein, belongs to a large family of related proteins distinguished by the presence of a highly conserved nucleotide binding domain (Walker box A). The XcpR protein is essential for the process of extracellular secretion and amino acid substitutions within the Walker A sequence result in inactive XcpR. The same mutations exert a dominant negative effect on protein secretion when expressed in wild-type bacteria. Transdominance of XcpR mutants suggests that this protein is involved in interactions with other components of the secretion machinery or that it functions as a multimer. In this study, the amino-terminal portion of the cI repressor protein of phage λ was used as a reporter of dimerization in Escherichia coli following fusion to full-length as well as a truncated form of XcpR. The cI–XcpR hybrid proteins were able to dimerize, as demonstrated by the immunity of bacteria expressing them to killing by λ phage. The full-length XcpR as well as several deletion mutants of XcpR were able to disrupt the dimerization of the chimeric cI–XcpR protein. The disruption of cI–XcpR dimers using the deletion mutants of XcpR, combined with the analysis of their dominant negative effects on protein secretion, was used to map the minimal dimerization domain of XcpR, which is located within an 85 amino acid region in its N-terminal domain. Taken together, the data presented in this paper suggest that the XcpR protein dimerizes via its N-terminus and that this dimerization is essential for extracellular protein secretion.     

Basolateral sorting of chloride channel 2 is mediated by interactions between a dileucine motif and the clathrin adaptor AP-1

In spite of the many key cellular functions of chloride channels, the mechanisms that mediate their subcellular localization are largely unknown. ClC-2 is a ubiquitous chloride channel usually localized to the basolateral domain of epithelia that regulates cell volume, ion transport, and acid–base balance; mice knocked out for ClC-2 are blind and sterile. Previous work suggested that CLC-2 is sorted basolaterally by TIFS⁸¹²LL, a dileucine motif in CLC-2''s C-terminal domain. However, our in silico modeling of ClC-2 suggested that this motif was buried within the channel''s dimerization interface and identified two cytoplasmically exposed dileucine motifs, ESMI⁶²³LL and QVVA⁶³⁵LL, as candidate sorting signals. Alanine mutagenesis and trafficking assays support a scenario in which ESMI⁶²³LL acts as the authentic basolateral signal of ClC-2. Silencing experiments and yeast three-hybrid assays demonstrated that both ubiquitous (AP-1A) and epithelium-specific (AP-1B) forms of the tetrameric clathrin adaptor AP-1 are capable of carrying out basolateral sorting of ClC-2 through interactions of ESMI⁶²³LL with a highly conserved pocket in their γ1-σ1A hemicomplex.     

The phosphatase PHLPP controls the cellular levels of protein kinase C

The life cycle of protein kinase C (PKC) is controlled by multiple phosphorylation and dephosphorylation steps. The maturation of PKC requires three ordered phosphorylations, one at the activation loop and two at COOH-terminal sites, the turn motif and the hydrophobic motif, to yield a stable and signaling-competent enzyme. Dephosphorylation of the enzyme leads to protein degradation. We have recently discovered a novel family of protein phosphatases named PH domain leucine-rich repeat protein phosphatase (PHLPP) whose members terminate Akt signaling by dephosphorylating the hydrophobic motif on Akt. Here we show that the two PHLPP isoforms, PHLPP1 and PHLPP2, also dephosphorylate the hydrophobic motif on PKC betaII, an event that shunts PKC to the detergent-insoluble fraction, effectively terminating its life cycle. Deletion mutagenesis reveals that the PH domain is necessary for the effective dephosphorylation of PKC betaII by PHLPP in cells, whereas the PDZ-binding motif, required for Akt regulation, is dispensable. The phorbol ester-mediated dephosphorylation of the hydrophobic site, but not the turn motif or activation loop, is insensitive to okadaic acid, consistent with PHLPP, a PP2C family member, controlling the hydrophobic site. In addition, knockdown of PHLPP expression reduces the rate of phorbol ester-triggered dephosphorylation of the hydrophobic motif, but not turn motif, of PKC alpha. Last, we show that depletion of PHLPP in colon cancer and normal breast epithelial cells results in an increase in conventional and novel PKC levels. These data reveal that PHLPP controls the cellular levels of PKC by specifically dephosphorylating the hydrophobic motif, thus destabilizing the enzyme and promoting its degradation.     

A PDZ-binding motif controls basolateral targeting of syndecan-1 along the biosynthetic pathway in polarized epithelial cells

The cell surface proteoglycan, syndecan-1, is essential for normal epithelial morphology and function. Syndecan-1 is selectively localized to the basolateral domain of polarized epithelial cells and interacts with cytosolic PDZ (PSD-95, discs large, ZO-1) domain-containing proteins. Here, we show that the polarity of syndecan-1 is determined by its type II PDZ-binding motif. Mutations within the PDZ-binding motif lead to the mislocalization of syndecan-1 to the apical surface. In contrast to previous examples, however, PDZ-binding motif-dependent polarity is not determined by retention at the basolateral surface but rather by polarized sorting prior to syndecan-1's arrival at the plasma membrane. Although none of the four known PDZ-binding partners of syndecan-1 appears to control basolateral localization, our results show that the PDZ-binding motif of syndecan-1 is decoded along the biosynthetic pathway establishing a potential role for PDZ-mediated interactions in polarized sorting.     

Association of ARVCF with zonula occludens (ZO)-1 and ZO-2: binding to PDZ-domain proteins and cell-cell adhesion regulate plasma membrane and nuclear localization of ARVCF

ARVCF, an armadillo-repeat protein of the p120(ctn) family, associates with classical cadherins and is present in adherens junctions, but its function is poorly understood. Here, we show that ARVCF interacts via a C-terminal PDZ-binding motif with zonula occludens (ZO)-1 and ZO-2. ARVCF and ZO-1 partially colocalize in the vicinity of the apical adhesion complex in polarized epithelial Madin-Darby canine kidney cells. ARVCF, ZO-1, and E-cadherin form a complex and are recruited to sites of initial cell-cell contact in sparse cell cultures. E-cadherin binding and plasma membrane localization of ARVCF require the PDZ-binding motif. Disruption of cell-cell adhesion releases ARVCF from the plasma membrane and an increased fraction of the protein localizes to the nucleus. Nuclear localization of ARVCF also requires the PDZ-binding motif and can be mediated by the PDZ domains of ZO-2. Thus, the interaction of ARVCF with distinct PDZ-domain proteins determines its subcellular localization. Interactions with ZO-1 and ZO-2, in particular, may mediate recruitment of ARVCF to the plasma membrane and the nucleus, respectively, possibly in response to cell-cell adhesion cues.     

Exploration of the TRIM Fold of MuRF1 Using EPR Reveals a Canonical Antiparallel Structure and Extended COS-Box

MuRF1 (TRIM63) is a RING-type E3 ubiquitin ligase with a predicted tripartite TRIM fold. TRIM proteins rely upon the correct placement of an N-terminal RING domain, with respect to C-terminal, specific substrate-binding domains. The TRIM domain organization is orchestrated by a central helical domain that forms an antiparallel coiled-coil motif and mediates the dimerization of the fold. MuRF1 has a reduced TRIM composition characterized by a lack of specific substrate binding domains, but contains in its helical domain a conserved sequence motif termed COS-box that has been speculated to fold independently into an α-hairpin. These characteristics had led to question whether MuRF1 adopts a canonical TRIM fold. Using a combination of electron paramagnetic resonance, on spin-labeled protein, and disulfide crosslinking, we show that TRIM63 follows the structural conservation of the TRIM dimerization domain, observed in other proteins. We also show that the COS-box motif folds back onto the dimerization coiled-coil motif, predictably forming a four-helical bundle at the center of the protein and emulating the architecture of canonical TRIMs.     

Leucine Zipper Motif Drives the Transmembrane Domain Dimerization of E-cadherin

E-cadherin is a transmembrane glycoprotein which is involved in the Ca²⁺-dependent cell–cell adhesion, and the adhesiveness is heavily dependent on the homodimerization of this molecule. Previous studies have shown that both the extracellular domain and cytoplasmic domain of E-cadherin contribute to its homodimerization. However, the roles of the transmembrane(TM) domain in the E-cadherin homodimerization have not been discussed in detail. In our experiments, SDS-PAGE showed higher molecular weight bands for the synthetic E-cadherin TM peptide, which indicated that the E-cadherin TM peptide is able to dimerize in the SDS micelle. The TOXCAT assay proved that the E-cadherin TM domain can form a moderate homo-oligomer in the Escherichia coli inner membrane. Furthermore, mutational analyses using the TOXCAT assays revealed that, instead of the common GxxxG dimerization motif, the leucine zipper motif is essential for the dimerization of the E-cadherin TM domain. Combining our experiment data and the computational simulation results, we provide insights for understanding the roles of the TM domain in the E-cadherin dimerization.     

Sorting nexin 27 interacts with the Cytohesin associated scaffolding protein (CASP) in lymphocytes

CASP is a small cytokine-inducible protein, primarily expressed in hematopoetic cells, which associates with members of the Cytohesin/ARNO family of guanine nucleotide-exchange factors. Cytohesins activate ARFs, a group of GTPases involved in vesicular initiation. Functionally, CASP is an adaptor protein containing a PDZ domain, a coiled-coil, and a potential carboxy terminal PDZ-binding motif that we sought to characterize here. Using GST pulldowns and mass spectrometry we identified the novel interaction of CASP and sorting nexin 27 (SNX27). In lymphocytes, CASP's PDZ-binding motif interacts with the PDZ domain of SNX27. This protein is a unique member of the sorting nexin family of proteins, a group generally involved in the endocytic and intracellular sorting machinery. Endogenous SNX27 and CASP co-localize at the early endosomal compartment in lymphocytes and also in transfection studies. These results suggest that endosomal SNX27 may recruit CASP to orchestrate intracellular trafficking and/or signaling complexes.     

Dimerization of CtIP may stabilize in vivo interactions with the Retinoblastoma-pocket domain

CtIP is a tumor suppressor that interacts with Retinoblastoma protein (Rb) to regulate the G1/S-phase transition of the cell cycle. Despite its large size (897 residues) CtIP has few known structured regions. Rather it contains several linear motifs that interact with known binding partners, including an LXCXE motif that binds the pocket domain of Rb-family proteins. This LXCXE motif lies at the C-terminus of the only known structured domain, an N-terminal coiled-coil dimerization domain (DD; residues 45-160). Yeast two-hybrid (Y2H) and GST-pulldown analyses showed that CtIP requires the LXCXE motif to bind the Rb-pocket. Although isothermal titration calorimetry data indicates that the LXCXE motif is the sole determinant of binding affinity for the Rb-pocket domain (K(A) approximately 10(6)M(-1)), Y2H data indicates that the DD is required to stabilize the interaction in vivo. Thus dimerization may increase the apparent stability of the proteins and/or the lifetime of the complexes.     

Ca2+-dependent monomer and dimer formation switches CAPRI Protein between Ras GTPase-activating protein (GAP) and RapGAP activities

CAPRI is a member of the GAP1 family of GTPase-activating proteins (GAPs) for small G proteins. It is known to function as an amplitude sensor for intracellular Ca(2+) levels stimulated by extracellular signals and has a catalytic domain with dual RasGAP and RapGAP activities. Here, we have investigated the mechanism that switches CAPRI between its two GAP activities. We demonstrate that CAPRI forms homodimers in vitro and in vivo in a Ca(2+)-dependent manner. The site required for dimerization was pinpointed by deletion and point mutations to a helix motif that forms a hydrophobic face in the extreme C-terminal tail of the CAPRI protein. Deletion of this helix motif abolished dimer formation but did not affect translocation of CAPRI to the plasma membrane upon cell stimulation with histamine. We found that dimeric and monomeric CAPRI coexist in cells and that the ratio of dimeric to monomeric CAPRI increases upon cell stimulation with histamine. Free Ca(2+) at physiologically relevant concentrations was both necessary and sufficient for dimer formation. Importantly, the monomeric and dimeric forms of CAPRI exhibited differential GAP activities in vivo; the wild-type form of CAPRI had stronger RapGAP activity than RasGAP activity, whereas a monomeric CAPRI mutant showed stronger RasGAP than RapGAP activity. These results demonstrate that CAPRI switches between its dual GAP roles by forming monomers or homodimers through a process regulated by Ca(2+). We propose that Ca(2+)-dependent dimerization of CAPRI may serve to coordinate Ras and Rap1 signaling pathways.