TGF beta 1 inhibits Ca2+-calcineurin-mediated activation in thymocytes

TGFbeta1 is a polypeptide growth modulatory and differentiation factor involved in many biological processes including immune homeostasis and self-tolerance. Tgfb1 knockout mice die around weaning age due to severe inflammation in most major organ systems, but the mechanism underlying this disease is not understood. In this study we demonstrate that Tgfb1(-/-) CD4(+)CD8(+) and CD4(+)CD8(-) thymocytes are hyperresponsive to receptor-mediated and receptor-independent mitogenic stimulation. A suboptimal concentration of ionomycin in the presence of PMA fully activates Tgfb1(-/-) thymocytes, whereas the inhibitors of Ca(2+) influx and calcineurin, EGTA and FK506, eliminate the hyperresponsiveness. Hence, the hypersensitivity of Tgfb1(-/-) thymocytes is due to a lowered threshold for Ca(2+)-dependent activation. Further, we demonstrate that the hypersensitivity of thymocytes results from the absence of TGFbeta1 and not from the inflammatory environment because the thymocytes are hyperresponsive in preinflammatory-stage Tgfb1(-/-) mice. Our results suggest for the first time that TGFbeta1 functions to inhibit aberrant T cell expansion by maintaining intracellular calcium concentration levels low enough to prevent a mitogenic response by Ca(2+)-independent stimulatory pathways alone. Consequently, TGFbeta1 prevents autoimmune disease through a Ca(2+) regulatory pathway that maintains the activation threshold above that inducible by self-MHC-TCR interactions.     

To (TGF)beta or not to (TGF)beta: fine-tuning of Smad signaling via post-translational modifications

Smad proteins are key signal transducers for the TGF-beta superfamily and are frequently inactivated in human cancers, yet the molecular basis of how their levels and activities are regulated remains unclear. Recent progress, discussed herein, illustrates the critical roles of Smad post-translational modifications in the cellular outcome to TGF-beta signaling.     

TGF beta signals through a heteromeric protein kinase receptor complex.

Transforming growth factor beta (TGF beta) binds with high affinity to the type II receptor, a transmembrane protein with a cytoplasmic serine/threonine kinase domain. We show that the type II receptor requires both its kinase activity and association with another TGF beta-binding protein, the type I receptor, to signal growth inhibition and early gene responses. Receptors I and II associate as interdependent components of a heteromeric complex: receptor I requires receptor II to bind TGF beta, and receptor II requires receptor I to signal. This mode of operation points to fundamental differences between this receptor and the protein-tyrosine kinase cytokine receptors.     

Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation

Ubiquitin-mediated proteolysis regulates the activity of diverse receptor systems. Here, we identify Smurf2, a C2-WW-HECT domain ubiquitin ligase and show that Smurf2 associates constitutively with Smad7. Smurf2 is nuclear, but binding to Smad7 induces export and recruitment to the activated TGF beta receptor, where it causes degradation of receptors and Smad7 via proteasomal and lysosomal pathways. IFN gamma, which stimulates expression of Smad7, induces Smad7-Smurf2 complex formation and increases TGF beta receptor turnover, which is stabilized by blocking Smad7 or Smurf2 expression. Furthermore, Smad7 mutants that interfere with recruitment of Smurf2 to the receptors are compromised in their inhibitory activity. These studies thus define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGF beta receptor for degradation.     

Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta2-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.     

Transforming growth factor type beta (TGF beta) inhibits G1 to S transition, but not activation of human B lymphocytes

Type beta transforming growth factor (TGF beta) is a polypeptide that may influence the growth of a variety of cell types in a positive or negative fashion. In this study we show that TGF beta markedly inhibits DNA synthesis in normal and neoplastic human B lymphocytes stimulated to proliferate with anti-immunoglobulins and B-cell growth factor (BCGF). Although TGF beta was needed during the initial 12 h of the culture to promote optimal inhibition, we found that it had little or no effect on several early to intermediate parameters of cell activation [( Ca2+]i increase, c-myc mRNA increase, cellular enlargement, RNA increase, and the increase in the expression of the 4F2 activation antigen). In contrast, TGF beta almost completely blocked the induction of transferrin receptor expression, which normally occurs in the late G1 phase of the cell cycle. Therefore, we conclude that TGF beta treatment leads to arrest of the cells in the middle to late G1 phase, prior to transferrin receptor expression.     

The insulin receptor activation process involves localized conformational changes.

The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation.     

Stimulation by TGF beta of chick embryo fibroblasts--inhibition by an IGFBP-3.

The multiple effects of TGF beta on cell proliferation are not well understood. Our results show that TGF beta was a good but transient mitogen for chick embryo fibroblasts. DNA synthesis was three- to fourfold increased, even at high concentrations of TGF beta. We did not show a bimodal effect. An inhibitor of cell growth, that inhibits 100% of stimulation induced by serum in CEF, was purified to homogeneity from medium conditioned by mouse 3T3 cells. This inhibitor has been shown to be an IGF-binding protein (mIGFBP-3). In the present work, this mIGFBP-3 inhibited the TGF beta stimulation by about 50%, while the stimulation induced by PDGF or insulin was not inhibited by mIGFBP-3. Furthermore, TGF beta stimulation, in the presence of a high concentration of insulin in conditions which would saturate IGF receptors, was not significantly inhibited by mIGFBP-3. All together these results suggest that a part of the mitogenic effect of TGF beta may be through increasing IGF secretion and eventually other growth factors such as PDGF (as suggested previously).     

A switch mechanism for G beta gamma activation of I(KACh)

G protein-gated inwardly rectifying potassium (GIRK) channels are a family of K(+)-selective ion channels that slow the firing rate of neurons and cardiac myocytes. GIRK channels are directly bound and activated by the G protein G beta gamma subunit. As heterotetramers, they comprise the GIRK1 and the GIRK2, -3, or -4 subunits. Here we show that GIRK1 but not the GIRK4 subunit is phosphorylated when heterologously expressed. We found also that phosphatase PP2A dephosphorylation of a protein in the excised patch abrogates channel activation by G beta gamma. Experiments with the truncated molecule demonstrated that the GIRK1 C-terminal is critical for both channel phosphorylation and channel regulation by protein phosphorylation, but the critical phosphorylation sites were not located on the C terminus. These data provide evidence for a novel switch mechanism in which protein phosphorylation enables G beta gamma gating of the channel complex.     

The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation: insight from structures of the prolactin receptor

The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has?a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane proximal domain of the human PRLR and find that the tryptophans of the motif adopt a T-stack conformation in the unbound state. By contrast, in the hormone bound state, a Trp/Arg-ladder is formed. The conformational change is hormone-dependent and influences the receptor-receptor dimerization site 3. In the constitutively active, breast cancer-related receptor mutant PRLR(I146L), we observed a stabilization of the dimeric state and a change in the dynamics of the motif. Here we demonstrate a structural link between the WSXWS motif, hormone binding, and receptor dimerization and propose it?as?a general mechanism for class 1 receptor activation.     

Binding of calmodulin to the D2-dopamine receptor reduces receptor signaling by arresting the G protein activation switch

Signaling by D(2)-dopamine receptors in neurons likely proceeds in the presence of Ca(2+) oscillations. We describe here the biochemical basis for a cross-talk between intracellular Ca(2+) and the D(2) receptor. By activation of calmodulin (CaM), Ca(2+) directly inhibits the D(2) receptor; this conclusion is based on the following observations: (i) The receptor contains a CaM-binding motif in the NH(2)-terminal end of the third loop, a domain involved in activating G(i/o). A peptide fragment encompassing this domain (D2N) bound dansylated CaM in a Ca(2+)-dependent manner (K(D) approximately 0.1 micrometer). (ii) Activation of purified Galpha(i1) by D2N, and D(2) receptor-promoted GTPgammaS (guanosine 5'-(3-O-thio)triphosphate) binding in membranes was suppressed by Ca(2+)/CaM (IC(50) approximately 0.1 micrometer). (iii) If Ca(2+) influx was elicited in D(2) receptor-expressing HEK293 cells, agonist-dependent inhibition of cAMP formation decreased. This effect was not seen with other G(i)-coupled receptors (A(1)-adenosine and Mel(1A)-melatonin receptor). (iv) The D(2) receptor was retained by immobilized CaM and radiolabeled CaM was co-immunoprecipitated with the receptor. Specifically, inhibition by CaM does not result from uncoupling the D(2) receptor from its cognate G protein(s); rather, CaM directly targets the D(2) receptor to block the receptor-operated G protein activation switch.     

TGF beta 2, LIF and FGF2 cooperate to induce nephrogenesis

The metanephric kidney develops from interactions between the epithelial ureteric bud and adjacent metanephric mesenchyme, which is induced by the bud to form the epithelia of the nephron. We have found that leukemia inhibitory factor (LIF) and transforming growth factor beta 2 (TGF beta 2) are secreted by inductive rat bud cells and cooperate to enhance and accelerate renal tubule formation in uninduced rat metanephric mesenchymal explants. LIF alone or TGF beta 2 with fibroblast growth factor 2 induced numerous tubules in isolated mesenchymes over an 8 day period, while (in combination) all three caused abundant tubule formation in 72 hours. Furthermore, neutralization of Wnt ligands with antagonist-secreted Frizzled-related protein 1 abrogated these responses and combinatorial cytokine/growth factor stimulation of explants augmented nuclear activation of Tcf1/Lef1, suggesting that LIF and TGF beta 2/FGF2 cooperate to regulate nephrogenesis through a common Wnt-dependent mechanism.     

Phosphorylation of nuclear protein is an early event in TGF beta 1 action.

Transforming growth factor beta (TGF beta) is a family of polypeptides that modulate growth and differentiation. TGF beta exerts its effects on target cells through interaction with specific cell surface receptors, but the signal transduction pathways are as yet largely unresolved. In this study we report that the growth inhibitory action of TGF beta on mink lung CCl 64 cells is associated with a rapid and transient phosphorylation of a number of nuclear proteins. In parallel, a transient expression of the immediate early gene jun B is observed. The expression of jun B can be inhibited by the protein kinase inhibitor H7 and can be augmented by the phosphatase inhibitor okadaic acid. Thus, protein phosphorylation can be a possible mechanism through which TGF beta 1 initiates early genomic responses.     

An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.