The role of syndecan cytoplasmic domain in basic fibroblast growth factor-dependent signal transduction.

To determine the role played by syndecan-4 cytoplasmic domain in the mediation of basic fibroblast growth factor (bFGF) signaling, immortalized human cells (ECV) were used to generate cell lines expressing constructs encoding full-length sequences for syndecan-4 (S4), syndecan-1 (S1), glypican-1 (G1), or chimeric proteins consisting of the ectoplasmic domain of glypican-1 linked to the transmembrane/cytoplasmic domain of syndecan-4 (G1-S4c) and the ectoplasmic domain of syndecan-4 linked to the glypican-1 glycosylphosphatidylinositol (GPI) anchor sequence (S4-GPI). Vector-transduced cells (VC) were used as controls. Expression of all these proteoglycans (except for the vector control) significantly increased cell-associated heparan sulfate mass and the number of low affinity bFGF-binding sites. However, in low serum medium, the addition of bFGF stimulated growth and migration of cells expressing S4 and G1-S4c constructs but not G1, S1, S4-GPI, or VC cells. Similar results were obtained using Matrigel growth assays. Mutations of heparan sulfate attachment sites on S4 construct abolished syndecan-4-dependent augmentation of bFGF responses. We conclude that cytoplasmic tail of syndecan-4 plays an important role in bFGF-mediated signal transduction.     

Model of cell signal transduction in a three-dimensional domain

Intracellular signalling molecules form pathways inside the cell. These pathways carry a signal to target proteins which results in cellular responses. We consider a spherical cell with two internal compartments containing localized activating enzymes where as deactivating enzymes are spread uniformly through out the cytosol. Two diffusible signalling molecules are activated at the compartments and later deactivated in the cytosol due to deactivating enzymes. The two signalling molecules are a single link in a cascade reaction and form a self regulated dynamical system involving positive and negative feedback. Using matched asymptotic expansions we obtain approximate solutions of the steady state diffusion equation with a linear decay rate. We obtain three-dimensional concentration profiles for the signalling molecules. We also investigate an extension of the above system which has multiple cascade reactions occurring between multiple signalling molecules. Numerically, we show that the speed of the signal is an increasing function of the number of links in the cascade.     

Yeast Kex1p is a Golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane

We have investigated the localization of Kex1p, a type I transmembrane carboxypeptidase involved in precursor processing within the yeast secretory pathway. Indirect immunofluorescence demonstrated the presence of Kex1p in a punctate organelle resembling the yeast Golgi apparatus as identified by Kex2p and Sec7p (Franzusoff, A., K. Redding, J. Crosby, R. S. Fuller, and R. Schekman. 1991. J. Cell Biol. 112:27-37). Glycosylation studies of Kex1p were consistent with a Golgi location, as Kex1p was progressively N-glycosylated in an MNN1-dependent manner. To address the basis of Kex1p targeting to the Golgi apparatus, we examined the cellular location of a series of carboxy-terminal truncations of the protein. The results indicate that a cytoplasmically exposed carboxy-terminal domain is required for retention of this membrane protein within the Golgi apparatus. Deletions of the retention region or overproduction of wild-type Kex1p led to mislocalization of Kex1p to the vacuolar membrane. This unexpected finding is discussed in terms of models involving either the vacuole as a default destination for membrane proteins, or by endocytosis to the vacuole following their default localization to the plasma membrane.     

Alterations in the cytoplasmic domain of CLCN2 result in altered gating kinetics.

Mutations in the human ClC-2 Cl(-) channel have been described to influence its function dramatically. To test for naturally occurring gene variants in a human population and their functionality, all 24 CLCN2 exons from a Central African population were sequenced. Six single amino acid exchanges in the intracellular N-terminus (P48R, R68H), in the pore domain (G199A), or in the intracellular C-terminus (R646Q, R725W, R747H) were identified at low frequency. Heterologous expression of these polymorphisms in Xenopus laevis oocytes demonstrated their functional significance as determined by two-electrode voltage-clamp. The polymorphisms R68H, R725W, and R747H exhibited faster voltage-stimulated gating as compared to the wild type channel, resulting in higher steady state currents of R725W. Probably due to decreased surface expression P48R, R68H, and R646Q mutants generated lower currents than the wild type channels. The inward currents of the mutated channels R725W, R747H, and G199A failed to increase during hypotonic swelling, a defect paralleled by impaired swelling-accelerated voltage-gating in one mutant (G199A). In conclusion, the Africans' gene pool comprises CLCN2 gene variants in the N-terminus, the C-terminus or the pore domain that affect surface expression and voltage- or cell-swelling-stimulated channel gating.     

Observation of signal transduction in three-dimensional domain swapping

p13suc1 (suc1) has two native states, a monomer and a domain-swapped dimer. The structure of each subunit in the dimer is identical to that of the monomer, except for the hinge loop that connects the exchanging domains. Here we find that single point mutations at sites throughout the protein and ligand binding both shift the position of the equilibrium between monomer and dimer. The hinge loop was shown previously to act as a loaded molecular spring that releases tension present in the monomer by adopting an alternative conformation in the dimer. The results here indicate that the release of strain propagates throughout the entire protein and alters the energetics of regions remote from the hinge. Our data illustrate how the signal conferred by the conformational change of a protein loop, elicited by domain swapping, ligand binding or mutation, can be sensed by a distant active site. This work highlights the potential role of strained loops in proteins: the energy they store can be used for both signal transduction and allostery, and they could steer the evolution of protein function. Finally, a structural mechanism for the role of suc1 as an adapter molecule is proposed.     

Identification of an essential region for growth signal transduction in the cytoplasmic domain of the human interleukin-4 receptor.

Interleukin-4 (IL-4) is a pleiotropic lymphokine which plays an important role in the immune system by regulating proliferation and differentiation of a wide variety of lymphoid and myeloid cells. These biological effects are manifested via binding of IL-4 to specific membrane-associated high affinity receptors. While the IL-4 receptor (IL-4R) cDNA expresses high affinity binding sites when transfected in COS7 cells, its intracellular domain lacks consensus motifs for known signal transducing molecules such as a tyrosine kinase. In this study, we use a DNA deletion approach to explore the mechanism of signal transduction utilized by the human IL-4R cDNA expressed in a murine pro-B cell line, Ba/F3 cells. Using this system, we have identified the critical region of the cytoplasmic domain of human IL-4R for human IL-4-induced transduction of a growth signal in these cells. Our data indicate that the critical region for signal transduction is located between amino acid residues 433-473 numbering from the carboxyl terminus. This region is highly conserved between mouse and human IL-4R but lacks homology with other cytokine receptors. Our studies additionally demonstrate that the cytoplasmic domain is not essential for forming high affinity IL-4-binding sites nor for ligand internalization.     

The language of SH2 domain interactions defines phosphotyrosine-mediated signal transduction

Natural languages arise in an unpremeditated fashion resulting in words and syntax as individual units of information content that combine in a manner that is both complex and contextual, yet intuitive to a native reader. In an analogous manner, protein interaction domains such as the Src Homology 2 (SH2) domain recognize and "read" the information contained within their cognate peptide ligands to determine highly selective protein-protein interactions that underpin much of cellular signal transduction. Herein, we discuss how contextual sequence information, which combines the use of permissive and non-permissive residues within a parent motif, is a defining feature of selective interactions across SH2 domains. Within a system that reads phosphotyrosine modifications this provides crucial information to distinguish preferred interactions. This review provides a structural and biochemical overview of SH2 domain binding to phosphotyrosine-containing peptide motifs and discusses how the diverse set of SH2 domains is able to differentiate phosphotyrosine ligands.     

Phosphorylation of TRAF2 inhibits binding to the CD40 cytoplasmic domain

TRAF2 is a signal transducing adaptor molecule which binds to the CD40 cytoplasmic domain. We have found that it is phosphorylated, predominantly on serine residues, when transiently overexpressed in 293 cells. The phosphorylation appears to be related to the signaling events that are activated by TRAF2 under these circumstances, since two nonfunctional mutants were found to be phosphorylated significantly less than the wild-type protein. Furthermore, the phosphorylation status of TRAF2 had significant effects on the ability of the protein to bind to CD40, as evidenced by our observations that the CD40 cytoplasmic domain interacted preferentially with underphosphorylated TRAF2 and that phosphatase treatment significantly enhanced the binding of TRAF2 to CD40. We conclude from these studies that the phosphorylation of TRAF2 is likely to play an important role in regulating signaling by virtue of its ability to influence the CD40-TRAF2 interaction.     

Functional and structural effects of seven-residue deletions on the coiled-coil cytoplasmic domain of a chemoreceptor

Chemoreceptors transmit signals from the environment to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors is strongly conserved and consists of a long alpha-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Changes in this domain during evolution are characterized by the presence of seven-residue insertions/deletions located symmetrically with respect to the hairpin turn, suggesting that specific interactions between the helices that form the hairpin are required for function. We assessed the impact of seven-residue deletions on the signalling ability and higher-order organization of the serine chemoreceptor from Escherichia coli. Our results indicate that symmetry alterations between the two branches of the cytoplasmic hairpin seriously compromise chemoreceptor function. Shorter functional versions of Tsr with symmetrical deletions form mixed trimers of dimers when coexpressed with Tar, the aspartate receptor of E. coli. However, Tar function in those cells is impaired, suggesting that the length difference between receptors introduces non-functional distortions into the chemoreceptor cluster. This observation is reinforced by the analysis of coexpression of Tar with chemoreceptors from Rhodobacter sphaeroides that naturally belong to a shorter-length class.     

The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways.

The function of the T cell antigen receptor (TCR) invariant chains, CD3 gamma, delta, epsilon, and zeta, is poorly understood. Evidence suggests that CD3 couples receptor ligand binding to intracellular signaling events. To examine the role of the CD3 zeta chain in TCR-mediated signal transduction, a chimeric protein linking the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of the zeta chain was constructed. The CD8/zeta chimera is expressed independently of the TCR and is capable of transducing signals that, by criteria of early and late activation, are indistinguishable from those generated by the intact TCR. These data indicate that CD8/zeta can activate the appropriate signal transduction pathways in the absence of CD3 gamma, delta, and epsilon, and suggest that the role of CD3 zeta is to couple the TCR to intracellular signal transduction mechanisms.     

FHA: a signal transduction domain with diverse specificity and function

The structure of the FHA domain of the Chfr mitotic checkpoint protein described in this issue of Structure represents one of only a few known structures of this newly discovered phosphoprotein binding domain with diverse function and specificity.     

Conformational changes in the integrin beta A domain provide a mechanism for signal transduction via hybrid domain movement

The ligand-binding head region of integrin beta subunits contains a von Willebrand factor type A domain (betaA). Ligand binding activity is regulated through conformational changes in betaA, and ligand recognition also causes conformational changes that are transduced from this domain. The molecular basis of signal transduction to and from betaA is uncertain. The epitopes of mAbs 15/7 and HUTS-4 lie in the beta(1) subunit hybrid domain, which is connected to the lower face of betaA. Changes in the expression of these epitopes are induced by conformational changes in betaA caused by divalent cations, function perturbing mAbs, or ligand recognition. Recombinant truncated alpha(5)beta(1) with a mutation L358A in the alpha7 helix of betaA has constitutively high expression of the 15/7 and HUTS-4 epitopes, mimics the conformation of the ligand-occupied receptor, and has high constitutive ligand binding activity. The epitopes of 15/7 and HUTS-4 map to a region of the hybrid domain that lies close to an interface with the alpha subunit. Taken together, these data suggest that the transduction of conformational changes through betaA involves shape shifting in the alpha7 helix region, which is linked to a swing of the hybrid domain away from the alpha subunit.     

Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems

Over the last two decades, a new and unifying concept of cellular organization has emerged in which modular protein-protein interactions provide an underlying framework through which signaling pathways are assembled and controlled. In this scheme, posttranslational modifications such as phosphorylation commonly exert their biological effects by regulating molecular interactions, exemplified by the ability of phosphotyrosine sites to bind selectively to SH2 domains. Although these interactions are rather simple in isolation, they can nonetheless be exploited to generate complex cellular systems. Here, I discuss experiments that have led to this view of dynamic cellular behavior and identify some current and future areas of interest in cell signaling.     

Phosphorylation-dependent down-modulation of CD4 requires a specific structure within the cytoplasmic domain of CD4.

Several structural features of the cytoplasmic domain of CD4 including phosphorylation of Ser-408 have been shown to be important in its endocytosis (Shin, J., Doyle, C., Yang, Z., Kappes, D., and Strominger, J.L. (1990) EMBO J. 9, 425-434). A series of cytoplasmic domain truncations have now indicated that the membrane proximal region of the cytoplasmic domain from Arg-396 to Lys-417 is sufficient for phorbol ester-induced internalization; this segment is predicted to be an alpha-helix. The severe impairment of endocytosis resulting from the mutation Ser-408 to Ala-408 is largely restored by a compensating mutation Ala-404 to Ser-404; phosphorylation of Ser-404 has been directly demonstrated. Furthermore, mutation of Met-407, Ile-410, Leu-413, or Leu-414 to a hydrophilic residue eliminated CD4 endocytosis as did domain truncation at Arg-412. Ser-408 was normally phosphorylated in all of these mutants, suggesting that other residues in this region, including the four hydrophobic amino acids, are also required for CD4 endocytosis. Immunofluorescence microscopy following staining of intact and permeabilized cells showed that all endocytosis defective mutants indeed remained on the cell surface even after phorbol ester treatment, while wild type CD4 was endocytosed and degraded in lysosomes. These data indicate that endocytosis requiring residues 397-417 and binding of lymphocyte tyrosine kinase at residues 417-429 are functions of independent segments of the cytoplasmic region and lead to a hypothesis regarding some features of the endocytic process.     

Evidence supporting a passive role for the insulin receptor transmembrane domain in insulin-dependent signal transduction

We previously have demonstrated that intramolecular interactions between alpha beta-alpha beta subunits are necessary for insulin-dependent activation of the protein kinase domain within a single alpha 2 beta 2 heterotetrameric insulin-receptor complex (Wilden, P. A., Morrison, B. D., and Pessin, J. E. (1989) Biochemistry 28, 785-792). To evaluate the role of the beta subunit transmembrane domain in the insulin-dependent signalling mechanism, mutant human insulin receptors containing a series of nested transmembrane domain deletions (amino acids 941-945) were generated and stable Chinese hamster ovary-transfected cell lines were obtained. In addition, a substitution of Val-938 for Glu (E/V938) similar to the oncogenic mutation found in the neu transmembrane domain was also introduced into the insulin receptor. Scatchard analysis of insulin binding to the stable Chinese hamster ovary cell lines expressing either wild type or mutant insulin receptors indicated equivalent receptor number (2-4 x 10(6)/cell) and similar high affinity binding constants (Kd 0.1-0.3 nM). 125I-Insulin affinity cross-linking demonstrated that all of the expressed insulin receptors were assembled and processed into alpha 2 beta 2 heterotetrameric complexes. Surprisingly, all the mutant insulin receptors retained insulin-stimulated autophosphorylation both in vivo and in vitro. Furthermore, endogenous substrate phosphorylation in vivo as well as insulin-stimulated thymidine incorporation into DNA were unaffected by the transmembrane domain mutations. These data demonstrate that marked structural alterations in the insulin receptor transmembrane domain do not interfere with insulin-dependent signal transduction.