Ras and Rap1: two highly related small GTPases with distinct function

The Ras-like family of small GTPases includes, among others, Ras, Rap1, R-ras, and Ral. The family is characterized by similarities in the effector domain. While the function of Ras is, at least in part, elucidated, little is known about other members of the family. Currently, much attention is focused on the small GTPase Rap1. Initially, this member was identified as a transformation suppressor protein able to revert the morphological phenotype of Ras-transformed fibroblasts. This has led to the hypothesis that Rap1 antagonizes Ras by interfering in Ras effector function. Recent analysis revealed that Rap1 is activated rapidly in response to activation of a variety of receptors. Rap1 activation is mediated by several second messengers, including calcium, diacylglycerol, and cAMP. Guanine nucleotide exchange factors (GEFs) have been identified that mediate these effects. The most interesting GEF is Epac, an exchange protein directly activated by cAMP, thus representing a novel cAMP-induced, protein kinase A-independent pathway. Furthermore, Rap1 is inactivated by specific GTPase-activating proteins (GAPs), one of which is regulated through an interaction with Galphai. While Ras and Rap1 may share some effector pathways, evidence is accumulating that Ras and Rap1 each regulate unique cellular processes in response to various extracellular ligands. For Rap1 these functions may include the control of cell morphology.     

Exclusion of CD45 from the T-cell receptor signaling area in antigen-stimulated T lymphocytes

T lymphocytes are activated by the engagement of their antigen receptors (TCRs) with complexes of peptide and major histocompatibility complex (MHC) molecules displayed on the cell surface of antigen-presenting cells (APCs) [1]. An unresolved question of antigen recognition by T cells is how TCR triggering actually occurs at the cell-cell contact area. We visualized T-cell-APC contact sites using confocal microscopy and three-dimensional reconstruction of z-sections. We show the rapid formation of a specialized signaling domain at the T-cell-APC contact site that is characterized by a broad and sustained area of tyrosine phosphorylation. The T-lymphocyte cell-surface molecule CD2 is rapidly recruited into this signaling domain, whereas TCRs progressively percolate from the entire T-cell surface into the phosphorylation area. Remarkably, the highly expressed phosphatase CD45 is excluded from the signaling domain. Our results indicate that physiological TCR triggering at the T-cell-APC contact site is the result of a localized alteration in the balance between cellular kinases and phosphatases. We therefore provide experimental evidence to support current models of T-cell activation based on CD45 exclusion from the TCR signaling area [2] [3] [4].     

CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling.

T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45- T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45- T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45- T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates.     

Differential roles of Rap1 and Rap2 small GTPases in neurite retraction and synapse elimination in hippocampal spiny neurons

The Rap family of small GTPases is implicated in the mechanisms of synaptic plasticity, particularly synaptic depression. Here we studied the role of Rap in neuronal morphogenesis and synaptic transmission in cultured neurons. Constitutively active Rap2 expressed in hippocampal pyramidal neurons caused decreased length and complexity of both axonal and dendritic branches. In addition, Rap2 caused loss of dendritic spines and spiny synapses, and an increase in filopodia-like protrusions and shaft synapses. These Rap2 morphological effects were absent in aspiny interneurons. In contrast, constitutively active Rap1 had no significant effect on axon or dendrite morphology. Dominant-negative Rap mutants increased dendrite length, indicating that endogenous Rap restrains dendritic outgrowth. The amplitude and frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-mediated miniature excitatory postsynaptic currents (mEPSCs) decreased in hippocampal neurons transfected with active Rap1 or Rap2, associated with reduced surface and total levels of AMPA receptor subunit GluR2. Finally, increasing synaptic activity with GABA(A) receptor antagonists counteracted Rap2's inhibitory effect on dendrite growth, and masked the effects of Rap1 and Rap2 on AMPA-mediated mEPSCs. Rap1 and Rap2 thus have overlapping but distinct actions that potentially link the inhibition of synaptic transmission with the retraction of axons and dendrites.     

Beta 1 integrin (CD29) expression on human postnatal T cell subsets defined by selective CD45 isoform expression

The integrin beta 1 (CD29) is a marker for total very late activation Ag integrins on cells, and exhibits considerable fluctuation in cell surface density at various stages of T cell development. We have analyzed beta 1 integrin expression on subsets of human thymus, and on T cells from healthy babies and children, in comparison to healthy adults aged 26 to 75. T cells from adult peripheral blood include a CD29-, a CD29lo, and a CD29hi set. Compared with adults, PBMC T cells from children have reduced numbers of both CD29lo and CD29hi subsets but equivalent numbers of CD29- T cells. The number of CD29hi T cells increases gradually with age, achieving adult levels only at about 26 yr of age; in aged adults (69 to 75 yr), nearly all T cells have a CD29hi phenotype. Most thymocytes and cord blood T cells, in contrast, have a single peak of CD29 staining that is intermediate to the two peaks seen in adults. Multi-negative progenitor and CD45RO- thymocytes (presumptive thymic generative line-age) are 98% CD29hi. Progenitor thymocytes and adult PBMC T cells express equivalent amounts of beta 1 and alpha 4, but progenitors are alpha 5hi, whereas PBMC T cells are alpha 5lo. T cells from children have reduced beta 1hi and alpha 5lo, but nearly comparable numbers of alpha 4hi. This suggests that the major very late activation Ag integrins during childhood may be alpha 5 beta 1 and alpha 4 complexed with an alternate beta chain. In children, the majority of CD29hi cells are also CD45RAhi, in contrast to the pattern in adults, in whom the majority of CD29hi T cells are CD45RA-. This suggests that in children, the main defense against infection may reside in the CD29hi45RAhi T cells, which have not yet made the transition to CD45RO and to bona fide memory status. The proliferative response to tetanus toxoid of 4- to 6-mo-old babies correlates with the number of CD29hi45RAhi T cells, suggesting that it derives at least in part from cells that do not express a "memory" phenotype. These observations show a pattern of alternating high and low density CD29 during T cell development, which is consistent with the idea that CD29 is a marker for functionally defined T cell sets. Analysis of the CD29 expression of CD29hi thymocytes developing in vitro supports this view. We suggest that the intensity of CD29 expression on a T cell varies, dependent upon the microenvironmental interactions required by a differentiating T cell.     

Mechanism of the spatio-temporal regulation of Ras and Rap1

Epidermal growth factor (EGF) activates Ras and Rap1 at distinct intracellular regions. Here, we explored the mechanism underlying this phenomenon. We originally noticed that in cells expressing Epac, a cAMP-dependent Rap1 GEF (guanine nucleotide exchange factor), cAMP activated Rap1 at the perinuclear region, as did EGF. However, in cells expressing e-GRF, a recombinant cAMP-responsive Ras GEF, cAMP activated Ras at the peripheral plasma membrane. Based on the uniform cytoplasmic expression of Epac and e-GRF, GEF did not appear to account for the non-uniform increase in the activities of Ras and Rap1. In contrast, when we used probes with reduced sensitivity to GTPase-activating proteins (GAPs), both Ras and Rap1 appeared to be activated uniformly in the EGF-stimulated cells. Furthermore, we calculated the local rate constants of GEFs and GAPs from the video images of Ras activation and found that GAP activity was higher at the central plasma membrane than the periphery. Thus we propose that GAP primarily dictates the spatial regulation of Ras family G proteins, whereas GEF primarily determines the timing of Ras activation.     

CD45 isoform expression on human neonatal T cells: expression and turnover of CD45 isoforms on neonatal versus adult T cells after activation.

Neonatal T cells are phenotypically similar to "naive" T cells from adult donors in the CD45 isoform expression. Despite the phenotypic similarity, large differences were found between neonatal and adult T cells when T cells were activated. After activation with PHA, adult CD45RA+ T cells began to express CD45RO and no loss of CD45RA expression had yet occurred at Day 3 post-stimulation. Three days after activation, CD45RA+ neonatal T cells also coexpressed CD45RO; however, in contrast to adult T cells, a marked loss of CD45RA was observed. We analyzed the rapid loss of CD45RA found in neonatal T cells. The de novo synthesis of CD45 isoforms in neonatal T cells was essentially the same as that in the adult T cells. Turnover of the CD45RA was very rapid in both resting adult and neonatal T cells. After activation with PHA, the turnover of CD45RA on adult T cells was decreased significantly, while the turnover of CD45RA on neonatal T cells was not changed after activation. Therefore, the regulation of CD45 isoform expression not only involves switches in alternative splicing, but also involves different regulation of turnover of these isoforms from the cell membrane.     

The delta subunit of retinal rod cGMP phosphodiesterase regulates the membrane association of Ras and Rap GTPases.

Post-translational modifications of GTPases from the Ras superfamily enable them to associate with membrane compartments where they exert their biological activities. However, no protein acting like Rho and Rab dissociation inhibitor (GDI) that regulate the membrane association of Rho and Rab GTPases has been described for Ras and closely related proteins. We report here that the delta subunit of retinal rod phosphodiesterase (PDEdelta) is able to interact with prenylated Ras and Rap proteins, and to solubilize them from membranes, independently of their nucleotide-bound (GDP or GTP) state. We show that PDEdelta exhibits striking structural similarities with RhoGDI, namely conservation of the Ig-like fold and presence of a series of hydrophobic residues which could act as in RhoGDI to sequester the prenyl group of its target proteins, thereby providing structural support for the biochemical activity of PDEdelta. We observe that the overexpression of PDEdelta interferes with Ras trafficking and propose that it may play a role in the process that delivers prenylated proteins from endomembranes, once they have undergone proteolysis and carboxymethylation, to the structures that ensure trafficking to their respective resident compartments.     

Effect of dexamethasone on T-cell receptor/CD3 expression

Glucocorticoid hormones (GCH) are anti-inflammatory and immunosuppressive agents that inhibit T-cell growth and activation. Since the T-cell receptor (TCR)/CD3 complex mediates T-lymphocyte activation, we studied the effect of in vitro dexamethasone (DEX), a synthetic GCH, on TCR/CD3 expression.DEX-treatment of a hybridoma T-cell line and normal un-transformed T-cell clones induced a decrease of the TCR/CD3 membrane expression after 4 days. After 4 weeks, TCR/CD3 was undetectable. However, the amount of mRNAs coding TCR/CD3 chains, including TCR, TCR, CD3, CD3 and CD3, as well as the amount of CD3 protein, a major component of the complex, were unaltered. By contrast, a decrease of the mRNAs deriving from the TCR gene locus, as well as of the TCR protein which is responsible for the membrane expression of the TCR/CD3 complex, was induced.These data suggest that the down-modulation of TCR expression is due to the diminution of TCR gene products in DEX-treated cells. (Mol Cell Biochem 167: 135-144, 1997)     

Voltage-gated ion channel Kv4.3 is associated with Rap guanine nucleotide exchange factors and regulates angiotensin receptor type 1 signaling to small G-protein Rap

The voltage-gated potassium channel Kv4.3 was coexpressed with its beta-subunit Kv channel-interacting protein 2 and the angiotensin type 1 receptor in HEK-293 cells. Proteomic analysis of proteins coimmunoprecipitated with Kv4.3 revealed that Kv4.3 is associated with Rap guanine nucleotide exchange factors MR-GEF and EPAC-1. Previously, we demonstrated that Kv4.3 interacts with the angiotensin type 1 receptor in HE293 cells and cardiac myocytes. On the basis of this, we investigated the angiotensin type 1 receptor signaling to small G-proteins Ras and Rap-1 in the presence and absence of the Kv4.3-Kv channel-interacting protein 2 macromolecular complex. Ras activation was not significantly affected by coexpression of Kv4.3 and Kv channel-interacting protein 2. Ras exhibited a rapid activation-inactivation pattern with maximum activity at 2.5 min after addition of angiotensin II. In contrast, activation of Rap-1 was affected dramatically by coexpression of Kv4.3 and Kv channel-interacting protein 2 with the angiotensin type 1 receptor. In the absence of Kv4.3 and Kv channel-interacting protein 2, stimulation of the angiotensin type 1 receptor resulted in steady activation of Rap-1 that reached a plateau 25 min after addition of angiotensin II. In the presence of Kv4.3 and Kv channel-interacting protein 2, Rap-1 reaches a maximum activity 2.5 min after addition of angiotensin II and then deactivates rapidly, demonstrating a pattern of activation similar to that of Ras. Our findings show that Kv4.3 regulates angiotensin type 1 receptor signaling to the small G-protein Rap-1.     

Small GTPases Rap1 and RhoA regulate superoxide formation by Rac1 GTPases activation during the phagocytosis of IgG-opsonized zymosans in macrophages

Phagocytic NADPH oxidase plays a critical role in superoxide generation in macrophage cells. Small GTPases, including Rac1 and Rac2, have been implicated in the regulation of NADPH oxidase activity. Rap1, which has no effect in a cell-free system of oxidase activation, recently has been proven to colocalize with cytochrome b(558). In addition, neutrophils from rap1A(-/-) mice reduce fMLP-stimulated superoxide production. Here, we tried to determine whether Rap1 also plays a role in the production of superoxide. IgG-opsonized zymosan (IOZ) particles treatment induced Rap1 activation and superoxide generation. Knock-down of Rap1 by si-Rap1 suppressed IOZ-induced superoxide formation. Sh-RhoA also reduced superoxide levels, but 8CPT-2Me-cAMP, an activator of Epac1 (a guanine nucleotide exchange factor (GEF) of Rap1), could recover the levels to the control value. When cells were stimulated by IOZ, Rap1 and Rac1 were translocated to the membrane, and then interacted with p22(phox). 8CPT-2Me-cAMP rescued sh-RhoA-induced reduction of the interaction between Rac1 and p22(phox), and enhanced lysophosphatidic acid (LPA)-induced increase of their interaction. Moreover, Rac1 activity was increased by both LPA and 8CPT-2Me-cAMP when treated with IOZ particles. Si-Vav2 impaired GTP-Rac1 levels in response to 8CPT-2Me-cAMP/IOZ. Phosphorylation of RhoA activates Rac1 in response to IOZ by the enhanced binding of phospho-RhoA to RhoGDI, leading to the release of Rac1 from the Rac1-RhoGDI complex. In conclusion, IOZ treatment induces Rap1 activation and phosphorylation of RhoA, which in turn cause Rac1 activation and promote Rac1 translocation to the membrane leading to binding with p22(phox) that activates NADPH oxidase and produces superoxide.     

Interactions of CD45-associated protein with the antigen receptor signaling machinery in T-lymphocytes.

CD45 is a transmembrane protein tyrosine phosphatase playing an essential role during T-cell activation. This function relates to the ability of CD45 to regulate p56(lck), a cytoplasmic protein tyrosine kinase necessary for T-cell antigen receptor (TCR) signaling. Previous studies have demonstrated that CD45 is constitutively associated in T-lymphocytes with a transmembrane molecule termed CD45-AP (or lymphocyte phosphatase-associated phosphoprotein). Even though the exact role of this polypeptide is unclear, recent analyses of mice lacking CD45-AP have indicated that its expression is also required for optimal T-cell activation. Herein, we wished to understand better the function of CD45-AP. The results of our studies showed that in T-cells, CD45-AP is part of a multimolecular complex that includes not only CD45, but also TCR, the CD4 and CD8 coreceptors, and p56(lck). The association of CD45-AP with TCR, CD4, and CD8 seemed to occur via the shared ability of these molecules to bind CD45. However, binding of CD45-AP to p56(lck) could take place in the absence of other lymphoid-specific components, suggesting that it can be direct. Structure-function analyses demonstrated that such an interaction was mediated by an acidic segment in the cytoplasmic region of CD45-AP and by the kinase domain of p56(lck). Interestingly, the ability of CD45-AP to interact with Lck in the absence of other lymphoid-specific molecules was proportional to the degree of catalytic activation of p56(lck). Together, these findings suggest that CD45-AP is an adaptor molecule involved in orchestrating interactions among components of the antigen receptor signaling machinery. Moreover, they raise the possibility that one of the functions of CD45-AP is to recognize activated Lck molecules and bring them into the vicinity of CD45.     

Synergy between CD40 ligation and IL-4 on fibroblast proliferation involves IL-4 receptor signaling.

Fibrosis can be an undesired consequence of activated cellular immune responses. The purpose of this work was to determine whether CD40 ligation and the pro-fibrotic cytokine IL-4 interact in regulating fibroblast proliferation and collagen production, and, if so, the mechanisms used. This study found that the combination of IL-4 and ligation of CD40 on the fibroblast cell surface had synergistic effects in stimulating fibroblast proliferation. In contrast, CD40 ligation negated the inhibitory effects of IFN-gamma on fibroblast proliferation. Western blotting analyses of fibroblast crude lysates revealed that a potential mechanism of the synergy between CD40 ligation and IL-4 was the phosphorylation of proteins at 130 kDa and, to a lesser degree, at 95, 85, and 75 kDa. Immunoprecipitation-Western blotting experiments showed that phosphorylation levels of IL-4Ralpha, Janus kinase 1, insulin receptor substrate 1, and insulin receptor substrate 2, factors with molecular mass close to the observed 130 kDa major phosphorylation band, increased in response to the combined CD40 ligation and IL-4 action. In contrast, there was no evidence that synergy was mediated by an increased expression of IL-4Ralpha chain, CD40, or the autocrine profibrotic cytokines IL-6 and TGF-beta. These findings suggest that CD40-CD40 ligand contacts between fibroblasts and cells secreting IL-4 may promote the profibrotic effects of IL-4 by affecting signal transduction and reducing the anti-fibrotic effects of IFN-gamma.     

CD1d and CD1c expression in human B cells is regulated by activation and retinoic acid receptor signaling

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity, we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo, activated and memory B cells expressed lower levels of CD1d compared with resting, naive, and marginal zone-like B cells. In vitro, CD1d was downregulated by all forms of B cell activation, leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone, whereas activation via the BCR significantly upregulated CD1c, particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes, an effect that was reversed by RARα agonists. However, BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells, in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity.     

The delta e13 isoform of the calcitonin receptor forms a six-transmembrane domain receptor with dominant-negative effects on receptor surface expression and signaling

The CTRdelta e13 splice variant of the rabbit calcitonin receptor, which lacks the 14 amino acids of the seventh transmembrane domain (TMD) that are encoded by exon 13, is poorly expressed on the cell surface, fails to mobilize intracellular calcium or activate Erk, and inhibits the cell surface expression of the full-length C1a isoform. Nuclear magnetic resonance- and fluorescence-activated cell sorter-based experiments showed that the residual seventh TMD of CTRdelta e13 fails to partition into the lipid bilayer, resulting in an extracellular C terminus. Truncating the receptor after residue 397 to delete the cytoplasmic tail resulted in reduced cell surface expression and an inability to mobilize intracellular calcium or activate Erk, but the truncated receptor did not inhibit C1a cell surface expression. In contrast, when the receptor was truncated after residue 374 to eliminate the entire seventh TMD domain and the C-terminal domain, the resulting receptor reduced the cell surface expression of C1a in a manner similar to that of CTRdelta e13. Thus, normal cell surface expression, mobilization of intracellular calcium, and Erk activation requires the cytoplasmic C-terminal tail of the CTR, whereas the absence of the seventh TMD in the transmembrane helical bundle causes the dominant-negative effect on the surface expression of C1a.