Grb10 has been described as a cellular partner of several receptor tyrosine kinases, including the insulin receptor (IR) and the insulin-like growth factor I (IGF-I) receptor (IGF-IR).Its cellular role is still unclear and a positive as well as an inhibitory role in mitogenesis depending on the cell context has been implicated. We have tested other mitogenic receptor tyrosine kinases as putative Grb10 partners and have identified the activated forms of platelet-derived growth factor (PDGF) receptor ␤ (PDGFR␤), hepatocyte growth factor receptor (Met), and fibroblast growth factor receptor as candidates. We have mapped Y771 as a PDFGR␤ site that is involved in the association with Grb10 via its SH2 domain. We have further investigated the putative role of Grb10 in mitogenesis with four independent experimental strategies and found that all consistently suggested a role as a positive, stimulatory signaling adaptor in normal fibroblasts.

Stress response is a fundamental form of behavioral and physiological plasticity. Here we describe how serotonin (5HT) governs stress behavior by regulating DAF-2 insulin/IGF-1 receptor signaling to the DAF-16/FOXO transcription factor at the nexus of development, metabolism, immunity, and stress responses in C. elegans. Serotonin-deficient tph-1 mutants, like daf-2 mutants, exhibit DAF-16 nuclear accumulation and constitutive physiological stress states. Exogenous 5HT and fluoxetine (Prozac) prevented DAF-16 nuclear accumulation in wild-type animals under stresses. Genetic analyses imply that DAF-2 is a downstream target of 5HT signaling and that distinct serotonergic neurons act through distinct 5HT receptors to influence distinct DAF-16-mediated stress responses. We suggest that modulation of FOXO by 5HT represents an ancient feature of stress physiology and that the C. elegans is a genetically tractable model that can be used to delineate the molecular mechanisms and drug actions linking 5HT, neuroendocrine signaling, immunity, and mitochondrial function.

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-␣ subunit (Gt␣) in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant Gt␣Q200L. An illumination threshold for the Gt␣ movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic Gt␣Q200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gt␣, the Gt␣Q200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of Gt␣Q200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gt␣, and apparently, do not require Gt␣ interaction with RGS9 and PDE6.Heterotrimeric GTP-binding proteins (G proteins) propagate a variety of hormonal and sensory signals from specific cell surface receptors to intracellular effectors (1-3). The visual transduction cascade in vertebrate photoreceptors has served for many years as a paradigm for G protein signaling. In rod photoreceptor cells, illuminated rhodopsin stimulates GTP-GDP exchange on the retinal G protein, transducin (Gt), 2 resulting in dissociation of Gt␣GTP from Gt␤␥ and rhodopsin. Gt␣ in the active GTP-bound conformation stimulates the effector enzyme, cGMP phosphodiesterase (PDE6), by displacing the inhibitory ␥-subunits (P␥) from the PDE6 catalytic core (PDE6␣␤). cGMP hydrolysis by active PDE6 results in closure of cGMP gated channels in the plasma membrane (4, 5). The turn-off phase of the visual signal is determined by reactions controlling the lifetimes of photoexcited rhodopsin (R*) and activated transducin. The catalytic function of R* is blocked by the rhodopsin-kinase mediated phosphorylation and the binding of arrestin to phosphorylated R* (6 -8). The lifetime of Gt␣GTP is controlled by intrinsic GTPase activity. Hydrolysis of GTP switches the Gt␣ molecule to the inactive GDP-bound conformation and allows reinhibition of PDE␣␤ by P␥. RGS9-1, a photoreceptor-specific member of the RGS (regulators of G protein signaling) family, in the complex with G␤5L acts as a GTPase-activating protein for transducin and thus is a major regulator of the turn-off kinetics of the visual signal (9 -11). The RGS9-1/G␤5L complex is anchored to disc membranes through the interaction with R9AP (RGS-9-1-anchor protein) that enhances the complex GAP acti...

Three cytoplasmic loops in the G protein-coupled receptor rhodopsin, C2, C3, and C4, have been implicated as key sites for binding and activation of the visual G protein transducin. Non-helical portions of the C2-and C3-loops and the cytoplasmic helix-8 from the C4 loop were targeted for a "gain-of-function" mutagenesis to identify rhodopsin residues critical for transducin activation. Mutant opsins with residues 140 -148 (C2-loop), 229 -244 (C3-loop), or 310 -320 (C4-loop) substituted by poly-Ala sequences of equivalent lengths served as templates for mutagenesis. The template mutants with polyAla substitutions in the C2-and C3-loops formed the 500-nm absorbing pigments but failed to activate transducin. Reverse substitutions of the Ala residues by rhodopsin residues have been generated in each of the templates. 313 and Met 317 produced a mutant pigment with the potency of transducin activation equal to that of the wild-type rhodopsin. Overall, our data support the role of the three cytoplasmic loops of rhodopsin and suggest that residues adjacent to the transmembrane helices are most important for transducin activation.The visual receptor, rhodopsin, is a prototypical G proteincoupled receptor (GPCR).1 It belongs to the class A GPCRs, the largest and the most studied within the GPCR superfamily (1-3). Rhodopsin is also the first and currently the only GPCR with a known crystal structure (4 -6). With a characteristic seven-transmembrane segment topography (helices H1-H7), a rhodopsin molecule forms three intradiscal (extracellular) (E1-E3) and three cytoplasmic loops (C1-C3). A fourth cytoplasmic loop (C4) results from the insertion of the palmitoyl groups attached to two cysteine residues within the C-terminal tail of rhodopsin, Cys 322 and Cys 323 . The key feature of the C4 loop is its largely helical structure (4). The C4 helix 8 (H8) stretches along the membrane plane through Cys 322 /Cys 323 in the direction almost perpendicular to H7. Deep within the helical transmembrane bundle lies the light-sensitive chromophore, 11-cisretinal, covalently linked via a protonated Schiff base to Lys 296 in H7. The ground state of rhodopsin (R) is stabilized by multiple contacts of the apoprotein with 11-cis-retinal and intermolecular interactions among transmembrane ␣-helices (4). The light-triggered 11-cis/trans-isomerization of retinal initiates a series of conformational changes in the rhodopsin molecule, leading to the transitional Meta II state (R*). In the Meta II conformation, the receptor cytoplasmic surface exposes sites for the interaction and activation of visual G protein, transducin (Gt) (2, 3). The light-induced rearrangements in rhodopsin have been extensively studied and include rigid body movements of at least H3, H6, and H7, which relay the conformational change to cytoplasmic loops C2, C3, and C4 (7-11). These loops are thought to be the key Gt interactions sites (12-16), although the role of C4 has been disputed (17, 18). Despite a large body of evidence for the involvement of C2, C3, and C4 in the...

The Nougaret form of dominant stationary night blindness is linked to a G38D mutation in the rod transducin-␣ subunit (T␣). In this study, we have examined the mechanism of Nougaret night blindness using transgenic mice expressing T␣G38D. The biochemical, electrophysiological, and vision-dependent behavioral analyses of the mouse model revealed a unique phenotype of reduced rod sensitivity, impaired activation, and slowed recovery of the phototransduction cascade. Two key deficiencies in T␣G38D function, its poor ability to activate PDE6 (cGMP phosphodiesterase) and decreased GTPase activity, are found to be the major mechanisms altering visual signaling in transgenic mice. Despite these defects, rod-mediated sensitivity in heterozygous mice is not decreased to the extent seen in heterozygous Nougaret patients.

Intraflagellar transport in cilia has been proposed as a crucial mediator of Hedgehog signal transduction during embryonic pattern formation in both vertebrates and invertebrates. Here, we show that the Hh receptor Patched-related factor DAF-6 and intraflagellar transport modulate serotonin production in Caenorhabditis elegans animals, by remodeling the architecture of dendritic cilia of a pair of ADF serotonergic chemosensory neurons. Wild-type animals under aversive environment drastically reduce DAF-6 expression in glialike cells surrounding the cilia of chemosensory neurons, resulting in cilium structural remodeling and upregulation of the serotoninbiosynthesis enzyme tryptophan hydroxylase tph-1 in the ADF neurons. These cellular and molecular modifications are reversed when the environment improves. Mutants of daf-6 or intraflagellar transport constitutively upregulate tph-1 expression. Epistasis analyses indicate that DAF-6/intraflagellar transport and the OCR-2/OSM-9 TRPV channel act in concert, regulating two layers of activation of tph-1 in the ADF neurons. The TRPV signaling turns on tph-1 expression under favorable and aversive conditions, whereas inactivation of DAF-6 by stress results in further upregulation of tph-1 independently of OCR-2/OSM-9 activity. Behavioral analyses suggest that serotonin facilitates larval animals resuming development when the environment improves. Our study revealed the cilium structure of serotonergic neurons as a trigger of regulated serotonin production, and demonstrated that a Hedgehog-related signaling component is dynamically regulated by environment and underscores neuroplasticity of serotonergic neurons in C. elegans under stress and stress recovery.

In Caenorhabditis elegans the Toll-interleukin receptor domain adaptor protein TIR-1 via a conserved mitogen-activated protein kinase (MAPK) signaling cascade induces innate immunity and upregulates serotonin (5-HT) biosynthesis gene tph-1 in a pair of ADF chemosensory neurons in response to infection. Here, we identify transcription factors downstream of the TIR-1 signaling pathway. We show that common transcription factors control the innate immunity and 5-HT biosynthesis. We demonstrate that a cysteine to tyrosine substitution in an ARM motif of the HEAT/Arm repeat region of the TIR-1 protein confers TIR-1 hyperactivation, leading to constitutive tph-1 upregulation in the ADF neurons, increased expression of intestinal antimicrobial genes, and enhanced resistance to killing by the human opportunistic pathogen Pseudomonas aeruginosa PA14. A forward genetic screen for suppressors of the hyperactive TIR-1 led to the identification of DAF-19, an ortholog of regulatory factor X (RFX) transcription factors that are required for human adaptive immunity. We show that DAF-19 concerts with ATF-7, a member of the activating transcription factor (ATF)/cAMP response element-binding B (CREB) family of transcription factors, to regulate tph-1 and antimicrobial genes, reminiscent of RFX-CREB interaction in human immune cells. daf-19 mutants display heightened susceptibility to killing by PA14. Remarkably, whereas the TIR-1-MAPK-DAF-19/ATF-7 pathway in the intestinal immunity is regulated by DKF-2/protein kinase D, we found that the regulation of tph-1 expression is independent of DKF-2 but requires UNC-43/Ca2+/calmodulin-dependent protein kinase (CaMK) II. Our results suggest that pathogenic cues trigger a common core-signaling pathway via tissue-specific mechanisms and demonstrate a novel role for RFX factors in neuronal and innate immune responses to infection.

Two putative orthologs to the human reduced folate carrier (hRFC), folt-1 and folt-2, which share a 40 and 31% identity, respectively, with the hRFC sequence, have been identified in the Caenorhabditis elegans genome. Functional characterization of the open reading frame of the putative folt-1 and folt-2 showed folt-1 to be a specific folate transporter. Transport of folate by folt-1 expressed in a heterologous expression system showed an acidic pH dependence, saturability (apparent Kmof 1.23 ± 0.18 μM), a similar degree of inhibition by reduced and substituted folate derivatives, sensitivity to the anti-inflammatory drug sulfasalazine (apparent Kiof 0.13 mM), and inhibition by anion transport inhibitors, e.g., DIDS. Knocking down (silencing) or knocking out the folt-1 gene led to a significant inhibition of folate uptake by intact living C. elegans. We also cloned the 5′-regulatory region of the folt-1 gene and confirmed promoter activity of the construct in vivo in living C. elegans. With the use of the transcriptional fusion construct (i.e., folt-1::GFP), the expression pattern of folt-1 in different tissues of living animal was found to be highest in the pharynx and intestine. Furthermore, folt-1::GFP expression was developmentally and adaptively regulated in vivo. These studies demonstrate for the first time the existence of a specialized folate uptake system in C. elegans that has similar characteristics to the folate uptake process of the human intestine. Thus C. elegans provides a genetically tractable model that can be used to study integrative aspects of the folate uptake process in the context of the whole animal level.