Ric-8: different cellular roles for a heterotrimeric G-protein GEF.

Signaling via heterotrimeric G-proteins is evoked by agonist-mediated stimulation of seven transmembrane spanning receptors (GPCRs). During the last decade it has become apparent that Gα subunits can be activated by receptor-independent mechanisms. Ric-8 belongs to a highly conserved protein family that regulates heterotrimeric G-protein function, acting as a non-canonical guanine nucleotide exchange factors (GEF) over a subset of Gα subunits. In this review we discuss the roles of Ric-8 in the regulation of diverse cell functions, emphasizing the contribution of its multiple domain protein structure in these diverse functions.

Cloning and spatiotemporal expression of RIC-8 in Xenopus embryogenesis.

RIC-8 is a highly conserved protein that promotes G protein signaling as it acts as a Guanine nucleotide Exchanging Factor (GEF) over a subset of Gα subunits. In invertebrates, RIC-8 plays crucial roles in synaptic transmission as well as in asymmetric cell division. As a first step to address further studies on RIC-8 function in vertebrates, here we have cloned a ric-8 gene from Xenopus tropicalis (xtric-8) and determined its spatiotemporal expression pattern throughout embryogenesis. The xtric-8 transcript is expressed maternally and zygotically and, as development proceeds, it shows a dynamic expression pattern. At early developmental stages, xtric-8 is expressed in the animal hemisphere, whereas its expression is later restricted to neural tissues, such as the neural tube and the brain, as well as in the eye and neural crest-derived structures, including those of the craniofacial region. Together, our findings suggest that RIC-8 functions are related to the development of the nervous system.

Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces its GDP/GTP exchange rate.

The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit.

The C2 cytosolic loop of adenylyl cyclase interacts with the activated form of G alpha s.

Using the yeast two-hybrid system, we studied the physical interaction between the complete C1 and C2 cytosolic domains of Xenopus laevis type 9 (xl9C1, xl9C2) and the C2 domain of rat type 6 (r6C2) adenylyl cyclase (AC). Heterodimerization between xl9C1 and xl9C2 and homodimerization between C2 (but not C1) domains was observed. Interaction between C2 and human G alpha s (hG alpha s) was also detected and was dependent on G alpha s activation. In contrast X. laevis G alpha s (xlG alpha s), which is 92% identical to hG alpha s, was unable to interact with any of the three AC cytosolic domains tested, corroborating previous findings that showed no effector activation. Through the construction of chimeras, we demonstrated that the amino-terminal half of xlG alpha s was responsible for the lack of interaction with AC. Chimeras between mouse G alpha i2 and G alpha s (N-mG alpha i2/C-G alpha s), that have previously shown to activate AC to a higher extent than wild-type G alpha s, also interacted with the C2 cytosolic domain and with a higher affinity. Interestingly, N-mG alpha i2/C-xlG alpha s chimera was not only able to interact with C2 but also with the C1 cytosolic domain.

Dual transduction signaling by a Xenopus muscarinic receptor: adenylyl cyclase inhibition and MAP kinase activation.

Using transient transfection of COS-7 and human embryonic kidney 293 cells, we studied the functional properties of a previously cloned muscarinic Xenopus receptor [Herrera et al. (1994): FEBS Lett 352:175-179] and its coupling to adenylyl cyclase (AC) and mitogen-activated protein kinase (MAPK) pathways. Expression of the Xenopus muscarinic receptor results in the inhibition of AC activity and activation of the MAPK pathway through a mechanism that involves a Pertussis-toxin-sensitive G-protein and the G beta gamma subunits. The signal transduction properties of this receptor are similar to the mammalian m2 and m4 muscarinic receptors. These results strongly support the idea that inhibition of AC and MAPK activation, signaled out from the muscarinic oocyte receptor, are involved in the oocyte maturation process.

Molecular cloning and expression of an adenylyl cyclase from Xenopus laevis oocytes.

We have cloned a cDNA that encodes a novel Xenopus laevis oocyte adenylyl cyclase (xlAC) using oligonucleotides against conserved mammalian adenylyl cyclase regions. The isolated cDNA is 4372 bp long with an open reading frame of 4065 nucleotides which encodes a protein of 1355 amino acids. Comparison of the deduced amino acid sequence with previously cloned mammalian adenylyl cyclases shows a low identity, 19.7% with type 2 rat adenylyl cyclase and 24.2% with type 4 rat adenylyl cyclase, indicating that this Xenopus isoform represents a new member of this protein family. Gene expression studies of the xlAC by reverse PCR showed that this gene is expressed in all oogenesis stages but not during early embryogenesis. Expression of the xlAC in COS-7 cells resulted in increased basal AC activity, that was stimulated by forskolin, Gpp(NH)p and aluminium fluoride, and was insensitive to calcium and calcium-calmodulin (Ca2(+)-CaM).

Site-directed mutants of the beta subunit of protein kinase CK2 demonstrate the important role of Pro-58.

The following amino acids of the Xenopus laevis beta subunit of protein kinase CK2 (casein kinase 2) were changed to alanine: Pro-58 (beta P-->A); Asp-59 and Glu-60 and Glu-61 (beta DEE-->AAA); His-151-153 (beta HHH-->AAA). The last 37 amino acids of the carboxyl end were deleted (beta delta 179-215). Stimulation of CK2 alpha catalytic subunit activity was measured with casein as substrate and the following relative activities were observed: beta P-->A > beta DEE-->AAA >>> beta WT > beta HHH-->AAA >>> beta delta 179-215. The beta DEE-->AAA and beta P-->A were similar to beta WT in reducing CD2 alpha binding to DNA but beta delta 179-215 was less active. The results indicate that both Pro-58 and the surrounding acidic cluster play roles in dampening the activation of CK2 alpha and that the carboxyl end of beta is involved in the interaction with CK2 alpha.

Activity of recombinant alpha and beta subunits of casein kinase II from Xenopus laevis.

Casein kinase II (CKII) is a ubiquitous protein kinase, found predominantly in cell nuclei, which has two subunits in a tetrameric alpha 2 beta 2 or alpha alpha' beta 2 conformation. The catalytic center is present in the alpha subunit which is active by itself while beta is a regulatory subunit that can greatly enhance the activity of alpha. The cDNA genes of Xenopus laevis coding for the alpha and beta subunits of CKII have been expressed in Escherichia coli and extensively purified. The recombinant subunits reconstitute a fully active holoenzyme when incubated in stoichiometric amounts. Mutations that change serines in positions 2 and 3 of the beta subunit for glycines completely eliminate the autophosphorylation site present in this subunit but do not significantly affect the capacity of beta to activate alpha. A fusion protein composed of glutathione transferase linked to the X. laevis CKII beta subunit can also activate alpha. This fusion protein binds to glutathione-agarose beads and can mediate the binding of the alpha subunit to this matrix. Conversely, the alpha subunit was found to bind to glass fiber filters in an active form that can still be activated by beta to an extent similar to that seen in solution. Using peptides containing tyrosine and glutamic acid as inhibitors of the activity of the isolated alpha subunit and of the holoenzyme, the effect of beta on the specificity of inhibition was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of metal ions on the activity of casein kinase II from Xenopus laevis.

Casein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant alpha and beta subunits of the X. laevis CKII, produced in E. coli from the cloned cDNA genes, were tested with different divalent metal ions. The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+. Optimal concentrations were 7-10 mM for Mg2+, 0.5-0.7 mM for Mn2+ and 1-2 mM for Co2+. In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence of Mg2+. The apparent Km values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+. Addition of Zn2+ (above 150 microM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and alpha subunit. Inhibition of the holoenzyme by 400 microM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the alpha subunit.

The cDNAs coding for the alpha- and beta-subunits of Xenopus laevis casein kinase II.

Using a lambda gt10 cDNA library obtained from Xenopus laevis oocytes and probes derived from the known sequences of the human and Drosophila genes, a cDNA coding for the alpha-subunit of the X. laevis casein kinase II was isolated. The coding sequence of this clone determines a polypeptide of 350 amino acids. The X. laevis sequence is 98% identical to the human and rat proteins in the first 323 amino acids. Using the polymerase chain reaction to generate a 370-nucleotide-long probe, it was possible to clone and sequence a cDNA of 900 nucleotides that coded for the X. laevis beta-subunit of casein kinase II. The derived protein sequence is 215 amino acids long and again shows an extraordinary degree of conservation with other species.

Oncogenic ras protein induces meiotic maturation of amphibian oocytes in the presence of protein synthesis inhibitors.

Microinjection of the activated ras oncogenic protein can induce the meiotic maturation of Xenopus laevis oocytes, a process that can also be triggered by progesterone or high concentrations of insulin. Cycloheximide and puromycin, well-known inhibitors of protein synthesis, block the maturation process induced by progesterone and insulin but do not affect the maturation caused by H-raslys12 protein microinjection. Theophylline, an inhibitor of cAMP phosphodiesterase that also affects oocyte protein synthesis, does cause a partial inhibition of ras protein-induced maturation. These findings indicate that ras protein acts on the oocyte maturation process at a point that is downstream of the protein synthesis requirement, a characteristic shared with the maturation promoting factor, an activity that appears in oocytes and mitotic cells at the onset of cell division.

Isolation and sequence determination of an active site peptide of rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase was inactivated by 2', 3'-dialdehyde ADP with the incorporation of one molecule of reagent per enzyme subunit. The inactivated protein was digested with trypsin after reduction and carboxymethylation. The labeled peptide was isolated by gel filtration and further purified by HPLC. The peptide was sequenced both by liquid-phase and gas-phase automatic Edman degradation. A 34-residue peptide was obtained. This peptide is identical to a tryptic peptide labeled with trinitrobenzenesulfonate, isolated and sequenced by Johnson et al. (Biochem. Biophys. Res. Commun. (1979) 90, 525-530) from bovine muscle pyruvate kinase. Available evidence suggests that dialdehyde ADP labels the enzyme at the same lysine in position 25 of the peptide, as found by Johnson et al. The high homology between the isolated peptide and regions of other pyruvate kinases from low to high eukaryotes supports the idea that this peptide is related to the enzyme active site.

Pyruvate kinase: studies on affinity labeling and active-site structure using the rabbit muscle enzyme.

Important advances have been made in recent years in the study of the structure of pyruvate kinase: the amino acid sequence of the enzymes from chicken muscle and yeast have been established and the three-dimensional structure of the cat muscle enzyme has been determined at 0.26 nm resolution. Work in our laboratory has shown that dialdehyde-ADP (oADP) can be used as an affinity label of rabbit muscle pyruvate kinase: if the enzyme is incubated with cold oADP in the presence of high ADP concentrations, dialyzed and then incubated with 14C-oADP, the enzyme inactivates and one mole of radioactive oADP incorporates per mole of enzyme subunit. A labeled peptide with a molecular weight of about 5900 has been purified from a tryptic digest of the modified enzyme. The first 26 residues of the peptide have been sequenced and this sequence is identical to a region in the chicken muscle enzyme and a peptide isolated from the bovine muscle enzyme specifically labeled with trinitrobenzenesulfonate. High homology is also found with a region of the yeast enzyme. All this suggests that the isolated peptide is part of the active site; the modified amino acid, probably a lysine, seems to be located in one of the alfa helices of domain A of the enzyme, according to the x-ray data.

Affinity labeling of rabbit muscle pyruvate kinase with dialdehyde-ADP.

Periodate-oxidized ADP (dialdehyde-ADP) inactivates rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and combines irreversibly to the enzyme. This inactivation is first-order with respect to dialdehyde-ADP and follows saturation kinetics, indicating that the enzyme first forms a reversible complex with the inactivator. Low Mg2+ concentrations stimulate the rate of inactivation, while higher concentrations have a protective effect. ADP and ATP, especially in the presence of Mg2+, protect very strongly against inactivation, while phosphoenolpyruvate and pyruvate are less effective. Dialdehyde-ADP is not a substrate, but acts as competitive inhibitor of ADP, with a KI of 4.5 mM. The analog has somewhat lower affinity to the enzyme than Mg-ADP, which has a Kd of 1.2 mM. Based on kinetic data, it is shown that one molecule of reagent must combine per enzyme active site in order to inactivate the enzyme. Incorporation of [14-C]dialdehyde-ADP to the enzyme and treatment of the data by the Tsou plot shows that 6-7 residues per subunit react with the modifier, two of them being essential for activity. From the evidence presented it is concluded: (1) dialdehyde-ADP behaves as an affinity label of rabbit muscle pyruvate kinase; (2) the inactivator binds probably to lysine residues at or near the active site, forming morpholine-like structures, and (3) the enzyme possesses two modifiable groups essential for activity, the reaction of one of them being sufficient to cause total loss in activity.