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Author(s): Alexandra Moers [1]; Bernhard Nieswandt [2]; Steffen Massberg [3]; Nina Wettschureck [1]; Sabine Gruner [2]; Ildiko Konrad [3]; Valerie Schulte [2]; Barsom Aktas [2]; Marie-Pierre Gratacap [1, 5]; Melvin [...]

RGS (regulator of G protein signaling) proteins containing the G protein [gamma]-like (GGL) domain (RGS6, RGS7, RGS9, and RGS11) interact with the fifth member of the G protein [beta]-subunit family, G[beta]5. This interaction is necessary for the stability of both the RGS protein and for G[beta]5. Consistent with this notion, we have found that elevation of RGS9-1 mRNA levels by transgene expression does not increase RGS9-1 protein level in the retina, suggesting that G[beta]5 levels may be limiting. To examine further the interactions of G[beta]5 and the GGL domain-containing RGS proteins, we inactivated the G[beta]5 gene. We found that the levels of GGL domain-containing RGS proteins in retinas and in striatum are eliminated or reduced drastically, whereas the levels of [G.sub.[gamma]2] and RGS4 proteins remain normal in the absence of G[beta]5. The homozygous G[beta]5 knockout (G[beta][5.sup.-/-]) mice derived from heterozygous knockout mating are runty and exhibit a high preweaning mortality rate. We concluded that complex formation between GGL domain-containing RGS proteins and the G[beta]5 protein is necessary to maintain their mutual stability in vivo. Furthermore, in the absence of G[beta]5 and all four RGS proteins that form protein complexes with G[beta]5, the animals that survive into adulthood are viable and have no gross defects in brain or retinal morphology.

Author(s): Participating investigators and scientists of the Alliance for Cellular Signaling ; Steering Committee:; Alfred G. Gilman [1]; Melvin I. Simon [6]; Henry R. Bourne [9]; Bruce A. Harris [13]; [...]

MrgA1 and MrgC11 belong to a recently identified family of orphan G-protein coupled receptors, called mrgs (mas-related genes). They are only expressed in a specific subset of sensory neurons that are known to detect painful stimuli. However, the precise physiological function of Mrg receptors and their underlying mechanisms of signal transduction are not known. We therefore have screened a series of neuropeptides against human embryonic kidney (HEK) 293 cells that stably express either MrgA1 or MrgC11 to identify ligands and/or agonists. MrgA1- or MrgC11-specific agonists stimulated dose-dependent increases in intracellular free [Ca.sup.2+] in a pertussis toxin-insensitive manner, but failed to alter basal or forskolin-stimulated levels of intracellular cAMP. Furthermore, studies using embryonic fibroblasts derived from various Ga protein knockout mice demonstrated that both the MrgA1 and MrgC11 receptors are coupled to the G[[alpha].sub.q/11] signaling pathway. Screening of neuropeptides identified surrogate agonists, most of these peptides included a common C-terminal -RF(Y)G or -RF(Y) amide motif. Structure-function studies suggest that endogenous ligands of Mrg receptors are likely to be RF(Y)G and/or RF(Y) amide-related peptides and that postprocessing of these peptides may serve to determine Mrg receptor-ligand specificity. The differences in ligand specificity also suggest functional diversity amongst the Mrg receptors.

G protein-coupled receptors (GPCRs), also know as mrgs, are expressed in nociceptive sensory neuron populations. Data suggest that mrgs may be involved in the regulation of nociceptor development and function and pain modulation.

Author(s): Ching-Kang Chen [1]; Marie E. Burns [2]; Wei He [3]; Theodore G. Wensel [3]; Denis A. Baylor [2]; Melvin I. Simon (corresponding author) [1] Timely deactivation of the α-subunit [...]

Elimination of excess climbing fiber (CF)Purkinje cell synapses during cerebellar development involves a signaling pathway that includes type 1 metabotropic glutamate receptor, G[Alpha]q, and the [Gamma] isoform of protein kinase C. To identify phospholipase C (PLC) isoforms involved in this process, we generated mice deficient in PLC[Beta]4, one of two major isoforms expressed in Purkinje cells. PLC[Beta]4 mutant mice are viable but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple CF innervation clearly is impaired in the rostral portion of the cerebellar vermis, in which PLC[Beta]4 mRNA is predominantly expressed. By contrast, CF synapse elimination is normal in the caudal cerebellum, in which low levels of PLC[Beta]4 mRNA but reciprocally high levels of PLC[Beta]3 mRNA are found. These results indicate that PLC[Beta]4 transduces signals that are required for CF synapse elimination in the rostral cerebellum.

The kinetics of the recoverin-rhodopsin kinase (RK) interaction was examined and the factors that affect the protein-protein relationship was studied using surface plasmon resonance. Results showed that the interaction was heavily influenced by Ca2+ and the rate of association and dissociation of recoverin-RK interaction occurs rapidly. Moreover, it was also observed that while autophosphorylation inhibits the ability of RK to bind with recoverin, ADP improves the interaction.

Arrestin binding does not seem to be rate limiting for recovery of the rod photoresponse in rods from transgenic mice in which one or both copies of the arrestin gene have been disrupted. When arrestin is not present, the flash response shows a very quick partial recovery. This is followed by a lengthy final phase. It seems that arrestin itself does not play a role in adaptation to background light. In -/- rods, the mechanisms of adaptation seemed to be normal, but they did not adapt normally because the long tails on the elementary responses pushed the response into saturation at very low light intensity.

Research shows that physiological platelet activators have no stimulatory affect on blood platelets from mice deficient in the alpha-subunit of the heterotrimeric guanine-nucleotide-binding protein. Extended bleeding times are noted. Thus, platelet activators need Galpha(sub q) to function properly. Galpha(sub i) or Beta gamma subunits of the heterotrimeric G proteins are not adequate substitutes for platelet activators. Evidence suggests that future drug development programs may use Galpha(sub q) to block platelet activation.

Expression of G protein-regulated phospholipase C (PLC) [Beta]4 in the retina, lateral geniculate nucleus, and superior colliculus implies that PLC [Beta]4 may play a role in the mammalian visual process. A mouse line that lacks PLC [Beta]4 was generated and the physiological significance of PLC [Beta]4 in murine visual function was investigated. Behavioral tests using a shuttle box demonstrated that the mice lacking PLC [Beta]4 were impaired in their visual processing abilities, whereas they showed no deficit in their auditory abilities. In addition, the PLC [Beta]4-null mice showed 4-fold reduction in the maximal amplitude of the rod a- and b-wave components of their electroretinograms relative to their littermate controls. However, recording from single rod photoreceptors did not reveal any significant differences between the PLC [Beta]4-null and wild-type littermates, nor were there any apparent differences in retinas examined with light microscopy. While the behavioral and electroretinographic results indicate that PLC [Beta]4 plays a significant role in mammalian visual signal processing, isolated rod recording shows little or no apparent deficit, suggesting that the effect of PLC [Beta]4 deficiency on the rod signaling pathway occurs at some stage after the initial phototransduction cascade and may require cell-cell interactions between rods and other retinal cells.

The in vivo and in vitro functions of a variety of CheZ mutants were characterized. To determine functional domains in the CheZ protein, mutants were constructed by random mutagenesis of the cheZ gene and a series of deletions were made. The map position and the in vivo and in vitro activity of the resulting gain- or loss-of-function mutant proteins define separate functional domains of the CheZ protein.

The cyclic AMP-dependent protein kinase (PKA) suppresses the activity of the phospholipase C (PLC)-beta-2 subunit which is activated by the G-beta-gamma protein in COS-7 cells. PKA has no effect on PLC isoforms that are activated by G-alpha. The suppression is due to the rapid phosphorylation of PLC-beta-2 serine amino acids. PKA and its isoforms have no suppressive effect on PLC isoforms.

The CheA1-233 fragment of the histidine autokinase CheA of Escherichia coli was studied using multidimensional heteronuclear nuclear magnetic resonance techniques. CheA is involved in chemotaxis in E. coli. The CheA1-233 fragment contains the CheY-binding domain and the phosphotransfer domain which are joined by a linker region. The results show that a flexible linker joins the CheY-binding domain and the phosphotransfer domain.

Open reading frames corresponding to cheA, cheW and cheY were discovered during sequencing of the Thermotoga maritima cheA gene clone. Expression of CheW and CheY in Escherichia coli revealed both proteins to be highly soluble and thermostable in the bacterium. The recombinant CheW protein was found to function as a small coupling protein in Escherichia coli. CheY, on the other hand, was found to remain active following purification from endogenous Escherichia coli protein.

Isothermal titration microcalorimetry experiments indicate the competitive behavior of response regulator proteins, CheY and the receptor methylesterase (CheB) to bind histidine kinase CheA in Escherichia coli. Phosphorylation decreases the binding affinity of CheY under physiological conditions. Mutations in the binding site of CheY reduces the association constant by 10-fold, and mutations in the active site produce lesser degrees of reduction.

A study of the phosphotransfer region, consisting of residues 1 to 134, of the histidine kinase CheA from Escherichia coli indicates that the domain has five alpha-helices named A to E, connected by turns. Nuclear magnetic resonance spectroscopy of the region reveals that the backbone has a compact structure and is rigid in helices and turns. The helix E interacts mainly with the helix D, and the three helices at the N-terminal end are unaffected by structural changes in helix E.

The goa-1 gene encoding the alpha subunit of the heterotrimeric guanosine triphosphate-binding protein (G protein) [G.sub.o] from Caenorhabditis elegans is expressed in most neurons, and in the muscles involved in egg laying and male mating. Reduction-of-function mutations in goa-1 caused a variety of behavioral defects including hyperactive movement, premature egg laying, and male impotence. Expression of the activated [G.sub.o] alpha subunit [Gα.sub.o]) in transgenic nematodes resulted in lethargic movement, delayed egg laying, and [reduced mating efficiency. Induced expression of activated [Gα.sub.o] in adults was sufficient to cause these phenotypes, indicating that [Gα.sub.o] mediates behavior through its role in neuronal function and the functioning of specialized muscles., The heterotrimeric G protein [G.sub.o] is abundantly expressed in mammalian and insect neurons and growth cones, suggesting that it is important for both neuronal development and function (1). Although G. [...]

Analysis of the ability of specific domains of phosducin to bind beta-gamma subunits of guanine nucleotide-binding proteins shows that the N-terminal 63-amino acid fragment is required for inhibition. A substitution of tryptophan-23 by valine blocks phosducin inhibitory function. The binding activity of phosducin is localized to the N-terminal fragments.

Cotransfection assay studies of the interaction between the beta and gamma subunits of G proteins reveal that a 173 amino acid-containing chimera between residues 210 and 293 in the C-terminal of the beta-1 subunit is essential for dimer formation with the gamma-1 subunit. This dimeric combination is crucial for several reactions of the G proteins.

In vivo competition experiments and in vitro binding assays demonstrate an interaction between the pleckstrin homology domain of Bruton tyrosine kinase (Btk) and the betagamma subunits of heterotrimeric guanine nucleotide-binding proteins. This interaction reveals that heterotrimeric G protein signaling pathways regulate the tyrosine kinase signaling pathway that is essential for the development and activity of B lymphocytes.

Autophosphorylating histidine kinase and response-regulator domains constitute the building blocks of two-component signaling systems. These systems use a unique phosphotransfer chemistry to regulate many aspects of bacterial physiology. Homologous systems are now being found in eukaryotes. Despite their common mechanism of phosphotransfer, the two-component systems display an extensive diversity in the arrangement of their domains, and flexibility in their roles in different signal transduction circuits.

Recombinant baculovirus were used to express a cDNA which encoded G16-alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein in Sf9 cells. Phospholipase C-beta-1 (PLC-beta-1) was stimulated by G16-alpha in membrane extracts of Sf9 cells when guanosine 5'-(gamma-thio)triphosphate, PLC-beta-1, PLC-beta-2 and PLC-beta-3 were stimulated by G16-alpha in a manner similar to that of G16-alpha. Stimulation of PLC-beta-1 and PLC-beta-3 intervened by G16-alpha were more than that of PLC-beta-2. PLC-gamma-1 or PLC-delta-1 were not stimulated by G16-alpha.

The regulation of the assemblage and mechanics of a signal transduction complex that controls chemotaxis in Escherichia coli, by using surface plasmon resonance biosensor technology, resulted in a quaternary complex consisting of a response regulator CheY, the histidine protein kinase CheA, a coupling protein CheW and a membrane-bound chemoreceptor Tar. The physical assessment of these interactions among the proteins in the single transduction system provides proof for a previous connecting interaction between the kinase and its substrate. This interaction is important for betterment of substrate specificity and stop 'crosstalk' with other systems, and for understanding transduction of intracellular messages by the signalling complexes.

The chemotaxis system of Escherichia coli comprises the histidine protein kinase CheA. The CheW coupling protein and the membrane-bound chemoreceptors control the autophosphorylation of CheA. The direction of flagellar rotation and the level of receptor adaptation are controlled by CheY and CheB proteins respectively, which are phosphorylated by CheA. The chemotactic response of the cells is thus regulated. The functional organization of the wild-type protein was defined by constructing and expressing genes encoding three polypeptide fragments of CheA. Surface plasmon resonance was used to map a stable binding site CheY by mapping a portion between the site of autophosphorylation and the kinase domain.

Researchers developed several phospholipase (PLC) beta 2 deletion mutants and chimeras having different parts of PLC beta and PLC beta 2 to find which parts of PLC beta 2 are needed for stimulation by G beta gamma or G alpha. The N-terminal portion of PLC beta 2 with amino acid sequence up to the end of the Y box is the area needed for stimulation by G beta gamma, and the C-terminal portion is the part having the amino acid sequence needed for stimulation by G alpha.

Important cell responses to hormones and other ligands are often mediated by the versatile G-protein system. The interaction of alpha-subunits of G proteins and a variety of effectors has been shown. Co-transfection assays can be used to show that a complex of the beta- and gamma-subunits of G (G-beta-gamma) specifically activate the beta-1 isoform of phospholipase. These results suggest that different effector pathways through a single receptor may be stimulated by G-alpha and G-beta-gamma subunits.

The mutant mouse rd allele corresponds to the retinal degeneration phenotype which has been associated with a reduction in cGMP phosphodiesterase (PDE) activity. The cDNA coding for the cGMP PDE beta subunit was isolated and fused to the 5' flanking region of the mouse rod opsin gene. This transgene construct was introduced into fertile one cell mouse embryos homozygous for the rd allele. One of the resulting transgenic lines showed complete rescue of the retinal degeneration phenotype, while a second line showed partial rescue. These results demonstrate that the cGMP PDE beta subunit is the site of the rd mutation.