Regulation of receptor protein-tyrosine phosphatase dimerization

Receptor protein-tyrosine phosphatases (RPTPs) are single membrane spanning proteins belonging to the family of PTPs that, together with the antagonistically acting protein-tyrosine kinases (PTKs), regulate the protein phosphotyrosine levels in cells. Protein-tyrosine phosphorylation is an important post-translational modification that has a major role in cell signaling by affecting protein-protein interactions and enzymatic activities. Increasing evidence indicates that RPTPs, like RPTKs, are regulated by dimerization. For RPTPalpha, we have shown that rotational coupling of the constitutive dimers in the cell membrane determines enzyme activity. Furthermore, oxidative stress, identified as an important second messenger during the past decade, is a regulator of rotational coupling of RPTPalpha dimers. In this review, we discuss the biochemical and cell biological techniques that we use to study the regulation of RPTPs by dimerization. These techniques include (co-) immunoprecipitation, RPTP activity assays, chemical and genetic cross-linking, detection of cell surface proteins by biotinylation, and analysis of RPTPalpha dimers, using conformation-sensitive antibody binding.     

Protein tyrosine phosphatase alpha (PTPalpha) and contactin form a novel neuronal receptor complex linked to the intracellular tyrosine kinase fyn

Glycosyl phosphatidylinositol (GPI)-linked receptors and receptor protein tyrosine phosphatases (RPTPs), both play key roles in nervous system development, although the molecular mechanisms are largely unknown. Despite lacking a transmembrane domain, GPI receptors can recruit intracellular src family tyrosine kinases to receptor complexes. Few ligands for the extracellular regions of RPTPs are known, relegating most to the status of orphan receptors. We demonstrate that PTPalpha, an RPTP that dephosphorylates and activates src family kinases, forms a novel membrane-spanning complex with the neuronal GPI-anchored receptor contactin. PTPalpha and contactin associate in a lateral (cis) complex mediated through the extracellular region of PTPalpha. This complex is stable to isolation from brain lysates or transfected cells through immunoprecipitation and to antibody-induced coclustering of PTPalpha and contactin within cells. This is the first demonstration of a receptor PTP in a cis configuration with another cell surface receptor, suggesting an additional mode for regulation of a PTP. The transmembrane and catalytic nature of PTPalpha indicate that it likely forms the transducing element of the complex, and we postulate that the role of contactin is to assemble a phosphorylation-competent system at the cell surface, conferring a dynamic signal transduction capability to the recognition element.     

Expression of receptor tyrosine phosphatases during development of the retinotectal projection of the chick

Receptor tyrosine kinases and receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including axon growth and guidance. Here, we focus on the possible roles of RPTPs in the developing avian retinotectal system. Using both in situ hybridization analysis and immunohistochemistry, we show for the first time that five RPTP genes--CRYPalpha, CRYP-2, PTPmu, PTPgamma, and PTPalpha--have different but overlapping expression patterns throughout the retina and the tectum. PTPalpha is restricted to Muller glia cells and radial glia of the tectum, indicating a possible function in controlling neuronal migration. PTPgamma expression is restricted to amacrine neurons. CRYPalpha and CRYP-2 mRNAs in contrast are expressed throughout the retinal ganglion cell layer from where axons grow out to their tectal targets. PTPmu is expressed in a subset of these ganglion cells. CRYPalpha, CRYP-2, and PTPmu proteins are also localized in growth cones of retinal ganglion cell axons and are present in defined laminae of the tectum. Thus, the spatial and temporal expression of three distinct RPTP subtypes--CRYPalpha, CRYP-2, and PTPmu--are consistent with the possibility of their involvement in axon growth and guidance of the retinotectal projection.     

Ectodomain shedding and intramembrane cleavage of mammalian Notch proteins is not regulated through oligomerization

Intramembrane cleaving proteases such as site 2 protease, gamma-secretase, and signal peptide peptidase hydrolyze peptide bonds within the transmembrane domain (TMD) of signaling molecules such as SREBP, Notch, and HLA-E, respectively. All three enzymes require a prior cleavage at the juxtamembrane region by another protease. It has been proposed that removing the extracellular domain allows dissociation of substrate TMD, held together by the extracellular domain or loop. Using gamma-secretase as a model intramembrane cleaving protease and Notch as a model substrate, we investigated whether activating and inactivating mutations in Notch modulate gamma-secretase cleavage through changes in oligomerization. We find that although the Notch epidermal growth factor repeats can promote dimer formation, most surface Notch molecules in mammalian cells are monomeric as are constitutively active or inactive Notch1 proteins. Using a bacterial assay for TM dimerization, we find that the isolated TMD of Notch and amyloid precursor protein self-associate and that mutations affecting Notch cleavage by gamma-secretase cleavage do not alter TMD dimerization. Our results indicate that ligand-induced reversal of controlled TMD dimerization by the Notch extracellular domain is unlikely to underlie the regulatory mechanism of intramembranous cleavage.     

Transmembrane helix dimerization: beyond the search for sequence motifs

Studies of the dimerization of transmembrane (TM) helices have been ongoing for many years now, and have provided clues to the fundamental principles behind membrane protein (MP) folding. Our understanding of TM helix dimerization has been dominated by the idea that sequence motifs, simple recognizable amino acid sequences that drive lateral interaction, can be used to explain and predict the lateral interactions between TM helices in membrane proteins. But as more and more unique interacting helices are characterized, it is becoming clear that the sequence motif paradigm is incomplete. Experimental evidence suggests that the search for sequence motifs, as mediators of TM helix dimerization, cannot solve the membrane protein folding problem alone. Here we review the current understanding in the field, as it has evolved from the paradigm of sequence motifs into a view in which the interactions between TM helices are much more complex. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Dimerization of tyrosine phosphatase PTPRO decreases its activity and ability to inactivate TrkC

Receptor-protein tyrosine phosphatases (RPTPs), like receptor tyrosine kinases, regulate neuronal differentiation. While receptor tyrosine kinases are dimerized and activated by extracellular ligands, the extent to which RPTPs dimerize, and the effects of dimerization on phosphatase activity, are poorly understood. We have examined a neuronal type III RPTP, PTPRO; we find that PTPRO can form dimers in living cells, and that disulfide linkages in PTPROs intracellular domain likely regulate dimerization. Dimerization of PTPROs transmembrane and intracellular domains, achieved by ligand binding to a chimeric fusion protein, decreases activity toward artificial peptides and toward a putative substrate, tropomyosin-related kinase C (TrkC). Dephosphorylation of TrkC by PTPRO may be physiologically relevant, as it is efficient, and TrkC and PTPRO can be co-precipitated from transfected cells. Inhibition of PTPROs phosphatase activity by dimerization is interesting, as dimerization of a related RPTP, CD148/PTPRJ, increases activity. Thus, our results suggest a complex relationship between dimerization and activity in type III RPTPs.     

The GxxxG-containing transmembrane domain of the CCK4 oncogene does not encode preferential self-interactions

The recently cloned colon carcinoma kinase 4 (CCK4) oncogene contains an evolutionarily conserved GxxxG motif in its single transmembrane domain (TMD). It has previously been suggested that this pairwise glycine motif may provide a strong driving force for transmembrane helix-helix interactions. Since CCK4 is thought to represent a new member of the receptor tyrosine kinase family, interactions between the TMDs may be important in receptor self-association and activation of signal transduction pathways. To determine whether this conserved CCK4 TMD can drive protein-protein interactions, we have carried out a thermodynamic study using the TMD expressed as a Staphylococcal nuclease (SN) fusion protein. Similar SN-TMD fusion proteins have been used to determine the sequence specificity and thermodynamics of transmembrane helix-helix interactions in a number of membrane proteins, including glycophorin A. Using sedimentation equilibrium in C14 betaine micelles, we discovered that the CCK4 TMD is unable to drive strong protein-protein interactions. At high protein/detergent ratios, the SN-CCK4 fusion protein will dimerize, but a stochastic model for protein association in micelles can explain the observed dimer population. For low-affinity interactions such as the one studied here, an understanding of this discrete stochastic distribution of membrane proteins in micelles is important for distinguishing between preferential and random self-interactions, which can both influence the oligomeric population. The lack of a thermodynamically meaningful self-association propensity for the CCK4 TMDs demonstrates that a GxxxG motif is not sufficient to drive transmembrane helix-helix interactions.     

Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.     

Transmembrane peptides as inhibitors of ErbB receptor signaling

Receptor tyrosine kinases have a single transmembrane (TM) segment that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, mutations within some of these receptors, and recent studies with the epidermal growth factor (EGF) and ErbB2 receptors have indicated that interactions between TM domains do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimerization and activation. One consequence of the importance of these interactions is that short hydrophobic peptides corresponding to these domains should act as specific inhibitors. To test this hypothesis, we constructed expression vectors encoding short fusion peptides encompassing native or mutated TM domains of the EGF, ErbB2, and insulin receptors. In human cell lines overexpressing the wild-type EGF receptor or ErbB2, we observed that the peptides are expressed at the cell surface and that they inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor. Identical results were obtained with peptides chemically synthesized. Mechanism of action involves inhibition of dimerization of the receptors as shown by the lack of effects of mutant nondimerizing sequences, completed by density centrifugation and covalent cross-linking experiments. Our findings stress the role of TM domain interactions in ErbB receptor function, and possibly for other single-spanning membrane proteins.     

Targeted disruption of the tyrosine phosphatase PTPalpha leads to constitutive downregulation of the kinases Src and Fyn.

A role for the receptor-like protein tyrosine phosphatase alpha (PTPalpha) in regulating the kinase activity of Src family members has been proposed because ectopic expression of PTPalpha enhances the dephosphorylation and activation of Src and Fyn [1] [2] [3]. We have generated mice lacking catalytically active PTPalpha to address the question of whether PTPalpha is a physiological activator of Src and Fyn, and to investigate its other potential functions in the context of the whole animal. Mice homozygous for the targeted PTPalpha allele (PTPalpha-/-) and lacking detectable PTPalpha protein exhibited no gross phenotypic defects. The kinase activities of Src and Fyn were significantly reduced in PTPalpha-/- mouse brain and primary embryonic fibroblasts, and this correlated with enhanced phosphorylation of the carboxy-terminal regulatory Tyr527 of Src in PTPalpha-/- mice. Thus, PTPalpha is a physiological positive regulator of the tyrosine kinases Src and Fyn. Increased tyrosine phosphorylation of several unidentified proteins was also apparent in PTPalpha-/- mouse brain lysates. These may be PTPalpha substrates or downstream signaling proteins. Taken together, the results indicate that PTPalpha has a dual function as a positive and negative regulator of tyrosine phosphorylation events, increasing phosphotyrosyl proteins through activation of Src and Fyn, and directly or indirectly removing tyrosine phosphate from other unidentified proteins.     

Post-translational membrane insertion of tail-anchored transmembrane EF-hand Ca2+ sensor calneurons requires the TRC40/Asna1 protein chaperone

Calneuron-1 and -2 are neuronal EF-hand-type calcium sensor proteins that are prominently targeted to trans-Golgi network membranes and impose a calcium threshold at the Golgi for phosphatidylinositol 4-OH kinase IIIβ activation and the regulated local synthesis of phospholipids that are crucial for TGN-to-plasma membrane trafficking. In this study, we show that calneurons are nonclassical type II tail-anchored proteins that are post-translationally inserted into the endoplasmic reticulum membrane via an association of a 23-amino acid-long transmembrane domain (TMD) with the TRC40/Asna1 chaperone complex. Following trafficking to the Golgi, calneurons are probably retained in the TGN because of the length of the TMD and phosphatidylinositol 4-phosphate lipid binding. Both calneurons rapidly self-associate in vitro and in vivo via their TMD and EF-hand containing the N terminus. Although dimerization and potentially multimerization precludes TRC40/Asna1 binding and thereby membrane insertion, we found no evidence for a cytosolic pool of calneurons and could demonstrate that self-association of calneurons is restricted to membrane-inserted protein. The dimerization properties and the fact that they, unlike every other EF-hand calmodulin-like Ca(2+) sensor, are always associated with membranes of the secretory pathway, including vesicles and plasma membrane, suggests a high degree of spatial segregation for physiological target interactions.     

High-Throughput Selection of Transmembrane Sequences That Enhance Receptor Tyrosine Kinase Activation

Dimerization is a critical requirement for the activation of the intracellular kinase domains of receptor tyrosine kinases (RTKs). The single transmembrane (TM) helices of RTKs contribute to dimerization, but the details are not well understood. Work with TM helices in various model systems has revealed a small number of specific dimerization sequence motifs, and it has been suggested that RTK dimerization is modulated by such motifs. Yet questions remain about the universality of these sequence motifs for RTK dimerization and about how TM domain dimerization in model systems relates to RTK activation in mammalian membranes. To investigate these questions, we designed a 3888-member combinatorial peptide library based on the TM domain of Neu (ErbB2) as a model RTK. The library contains many closely related, Neu-like sequences, including thousands of sequences with known dimerization motifs. We used an SDS-PAGE-based screen to select peptides that dimerize better than the native Neu sequence, and we assayed the activation of chimeric Neu receptors in mammalian cells with TM sequences selected in the screen. Despite the very high abundance of known dimerization motifs in the library, only a very few dimerizing sequences were identified by SDS-PAGE. About half of those sequences activated the Neu kinase significantly more than did the wild-type TM sequence. This work furthers our knowledge about the requirements for membrane protein interactions and the requirements for RTK activation in cells.     

Genetic selection for and molecular dynamic modeling of a protein transmembrane domain multimerization motif from a random Escherichia coli genomic library

In order to identify new transmembrane helix packing motifs in naturally occurring proteins, we have selected transmembrane domains from a library of random Escherichia coli genomic DNA fragments and screened them for homomultimerization via their abilities to dimerize the bacteriophage lambda cI repressor DNA-binding domain. Sequences were isolated using a modified lambda cI headpiece dimerization assay system, which was shown previously to measure transmembrane helix-helix association in the E. coli inner membrane. Screening resulted in the identification of several novel sequences that appear to mediate helix-helix interactions. One sequence, representing the predicted sixth transmembrane domain (TM6) of the E. coli protein YjiO, was chosen for further analysis. Using site-directed mutagenesis and molecular dynamics, a small set of models for YjiO TM6 multimerization interface interactions were generated. This work demonstrates the utility of combining in vivo genetic tools with computational systems for understanding membrane protein structure and assembly.     

The single transmembrane domains of ErbB receptors self-associate in cell membranes.

Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.     

The crystal structure of human receptor protein tyrosine phosphatase kappa phosphatase domain 1

The receptor-type protein tyrosine phosphatases (RPTPs) are integral membrane proteins composed of extracellular adhesion molecule-like domains, a single transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain consists of tandem PTP domains, of which the D1 domain is enzymatically active. RPTPkappa is a member of the R2A/IIb subfamily of RPTPs along with RPTPmu, RPTPrho, and RPTPlambda. Here, we have determined the crystal structure of catalytically active, monomeric D1 domain of RPTPkappa at 1.9 A. Structural comparison with other PTP family members indicates an overall classical PTP architecture of twisted mixed beta-sheets flanked by alpha-helices, in which the catalytically important WPD loop is in an unhindered open conformation. Though the residues forming the dimeric interface in the RPTPmu structure are all conserved, they are not involved in the protein-protein interaction in RPTPkappa. The N-terminal beta-strand, formed by betax association with betay, is conserved only in RPTPs but not in cytosolic PTPs, and this feature is conserved in the RPTPkappa structure forming a beta-strand. Analytical ultracentrifugation studies show that the presence of reducing agents and higher ionic strength are necessary to maintain RPTPkappa as a monomer. In this family the crystal structure of catalytically active RPTPmu D1 was solved as a dimer, but the dimerization was proposed to be a consequence of crystallization since the protein was monomeric in solution. In agreement, we show that RPTPkappa is monomeric in solution and crystal structure.     

Polar/Ionizable Residues in Transmembrane Segments: Effects on Helix-Helix Packing

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes.     

Evidence for New Homotypic and Heterotypic Interactions between Transmembrane Helices of Proteins Involved in Receptor Tyrosine Kinase and Neuropilin Signaling

Signaling in eukaryotic cells frequently relies on dynamic interactions of single-pass membrane receptors involving their transmembrane (TM) domains. To search for new such interactions, we have developed a bacterial two-hybrid system to screen for both homotypic and heterotypic interactions between TM helices. We have explored the dimerization of TM domains from 16 proteins involved in both receptor tyrosine kinase and neuropilin signaling. This study has revealed several new interactions. We found that the TM domain of Mucin-4, a putative intramembrane ligand for erbB2, dimerizes not only with erbB2 but also with all four members of the erbB family. In the Neuropilin/Plexin family of receptors, we showed that the TM domains of Neuropilins 1 and 2 dimerize with themselves and also with Plexin-A1, Plexin-B1, and L1CAM, but we were unable to observe interactions with several other TM domains notably those of members of the VEGF receptor family. The potentially important Neuropilin 1/Plexin-A1 interaction was confirmed using a surface plasmon resonance assay. This work shows that TM domain interactions can be highly specific. Exploring further the propensities of TM helix–helix association in cell membrane should have important practical implications related to our understanding of the structure–function of bitopic proteins' assembly and subsequent function, especially in the regulation of signal transduction.     

Dimerization of protein tyrosine phosphatase sigma governs both ligand binding and isoform specificity

Signaling through receptor protein tyrosine phosphatases (RPTPs) can influence diverse processes, including axon development, lymphocyte activation, and cell motility. The molecular regulation of these enzymes, however, is still poorly understood. In particular, it is not known if, or how, the dimerization state of RPTPs is related to the binding of extracellular ligands. Protein tyrosine phosphatase sigma (PTPsigma) is an RPTP with major isoforms that differ in their complements of fibronectin type III domains and in their ligand-binding specificities. In this study, we show that PTPsigma forms homodimers in the cell, interacting at least in part through the transmembrane region. Using this knowledge, we provide the first evidence that PTPsigma ectodomains must be presented as dimers in order to bind heterophilic ligands. We also provide evidence of how alternative use of fibronectin type III domain complements in two major isoforms of PTPsigma can alter the ligand binding specificities of PTPsigma ectodomains. The data suggest that the alternative domains function largely to change the rotational conformations of the amino-terminal ligand binding sites of the ectodomain dimers, thus imparting novel ligand binding properties. These findings have important implications for our understanding of how heterophilic ligands interact with, and potentially regulate, RPTPs.     

The Pathogenic A391E Mutation in FGFR3 Induces a Structural Change in the Transmembrane Domain Dimer

Fibroblast growth factor receptor 3 (FGFR3) is a single-pass membrane protein and a member of the receptor tyrosine kinase family of proteins that is involved in the regulation of skeletal growth and development. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain and the transmembrane domain (TMD). Previous work with the isolated FGFR3 TMD has shown that it has the ability to dimerize. Clinical and genetic studies have also correlated mutations in the TMD with a variety of skeletal and cranial dysplasias and cancer. Although the structures of the extracellular and cytosolic domains of FGFR3 have been solved, the structure of the TMD dimer is still unknown. Furthermore, very little is known regarding the effects of pathogenic mutations on the TMD dimer structure. We, therefore, carried out ToxR activity assays to determine the role of the SmXXXSm motif in the dimerization of the FGFR3 TMD. This motif has been shown to drive the association of many transmembrane proteins. Our results indicate that the interaction between wild-type FGFR3 TMDs is not mediated by two adjacent SmXXXSm motifs. In contrast, studies using the TMD carrying the pathogenic A391E mutation suggest that the motifs play a role in the dimerization of the mutant TMD. Based on these observations, here we report a new mechanistic model in which the pathogenic A391E mutation induces a structural change that leads to the formation of a more stable dimer.     

Sequence-specific dimerization of the transmembrane domain of the "BH3-only" protein BNIP3 in membranes and detergent

Mitochondria-mediated apoptosis is regulated by proteins of the Bcl-2 superfamily, most of which contain a C-terminal hydrophobic domain that plays a role in membrane targeting. Experiments with BNIP3 have implicated the transmembrane (TM) domain in its proapoptotic function, homodimerization, and interactions with Bcl-2 and Bcl-xL. We show that the BNIP3 TM domain self-associates strongly in Escherichia coli cell membranes and causes reversible dimerization of a soluble protein in the detergent SDS when expressed as an in-frame fusion. Limited mutational analysis identifies specific residues that are critical for BNIP3 TM self-association in membranes, and these residues are also important for dimerization in SDS micelles, suggesting that the self-association observed in membranes is preserved in detergent. The effects of sequence changes at positions Ala176 and Gly180 suggest that the BNIP3 TM domain associates using a variant of the GXXXG motif previously shown to be important in the dimerization of glycophorin A. The importance of residue His173 in BNIP3 TM domain dimerization indicates that polar residues, which have been implicated in self-association of model TM peptides, can act in concert with the AXXXG motif to stabilize TM domain interactions. Our results demonstrate that the hydrophobic C-terminal TM domain of the pro-apoptotic BNIP3 protein dimerizes tightly in lipidic environments, and that this association has a strong sequence dependence but is independent of the identity of flanking regions. Thus, the transmembrane domain represents another region of the Bcl-2 superfamily of proteins that is capable of mediating strong and specific protein-protein interactions.