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.

Chemical modification and site-directed mutagenesis of human liver arginase: evidence that the imidazole group of histidine-141 is not involved in substrate binding.

Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were photoinactivated by Rose bengal, and protection was afforded by the competitive inhibitor l-lysine. The dissociation constant for the enzyme-protector complex was essentially equal to the corresponding K(i) value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wild-type activity, with no changes in K(m) for arginine or K(i) for l-lysine or l-ornithine. The subunit composition of active enzyme was not altered by mutation, but the mutant H141F was markedly more sensitive to trypsin inactivation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DEPC were also insensitive to photoinactivation. We conclude that His141, which is the target for both inactivating procedures, is not involved in substrate binding, but plays a critical, albeit not essential role in the hydrolysis of enzyme-bound substrate.

Evidence that histidine-163 is critical for catalytic activity, but not for substrate binding to Escherichia coli agmatinase.

Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was inactivated by diethyl pyrocarbonate (DEPC) and illumination in the presence of Rose bengal. Protection against photoinactivation was afforded by the product putrescine, and the dissociation constant of the enzyme-protector complex (12 mM) was essentially equal to the K(i) value for this compound acting as a competitive inhibitor of agmatine hydrolysis. Upon mutation of His163 by phenylalanine, the agmatinase activity was reduced to 3-5% of wild-type activity, without any change in K(m) for agmatine or K(i) for putrescine inhibition. The mutant was insensitive to DEPC and dye-sensitized inactivations. We conclude that His163 plays an important role in the catalytic function of agmatinase, but it is not directly involved in substrate binding.

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).

Partial DNA sequence of a beta-lactamase produced by a Shigella flexneri strain.

A probe was constructed by radioactive labelling, and enzymatically a DNA fragment of plasmid pMAM-1, which codes for a beta-lactamase in Shigella flexneri UCSM 129, was obtained by amplification of a small part of the gene using the polymerase chain reaction technique (PCR). Since previous published work indicated that this beta-lactamase was of the TEM type, the primers used to amplify the gene were two highly conserved DNA regions in all TEM beta-lactamases. A 500 bp DNA probe was obtained which, by hybridization assays, facilitated the identification of restriction fragments of the plasmid containing the beta-lactamase gene. Two DNA fragments were sequenced by the Sanger method adapted to the PCR technique, and the sequence obtained showed a 100% homology with beta-lactamases TEM-1, TEM-2, TEM-13 and TEM-19. An intragenic restriction site, detected for Pst I, suggested that there is only one copy of the beta-lactamase gene per plasmid copy.

Cloning of a Xenopus laevis muscarinic receptor encoded by an intronless gene.

The Xenopus laevis oocyte has endogenous sites that bind muscarinic agonists, which have been pharmacologically characterized as M3 and/or M1 receptor subtypes. In order to define the molecular identify of the receptor protein we have analyzed a Xenopus oocyte cDNA library and cloned a 2.9 kb cDNA fragment encoding a muscarinic receptor (xMR). The deduced amino acid sequence reveals a protein of 484 residues with an apparent molecular weight of 54,188 Da. Amino acid comparison with previously cloned mammalian muscarinic receptors showed a 78% identity with the human m4 subtype, presenting at the same time clustered differences within the amino-terminal region and third intracellular loop Genomic Southern analysis displayed the presence of one main gene belonging to this subtype, and the PCR analysis revealed an intronless gene.

Human-Xenopus chimeras of Gs alpha reveal a new region important for its activation of adenylyl cyclase.

G proteins are heterotrimeric GTPases that play a key role in signal transduction. The alpha subunit of Gs bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X. laevis cDNA clones that apparently code for Gs alpha subunits are 92% identical to those found in the short form of human Gs alpha. Despite this high homology, the X. laevis Gs alpha clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc- membranes in contrast with human Gs alpha similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human Gs alpha. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus Gs alpha. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTP gamma and isoproterenol. The results obtained here clearly indicate that the G alpha sequence that extends from amino acid 70 to 140, is important for the functionality of human Gs alpha in activating adenylyl cyclase.

Xenopus laevis oocyte G alpha subunits mRNAs. Detection and quantitation during oogenesis and early embryogenesis by competitive reverse PCR.

The expression of mRNAs coding for different Xenopus laevis oocyte G alpha subunits was analyzed by the PCR technique. Using the nucleotide sequences of five previously cloned cDNAs for oocyte G alpha subunits [FEBS Lett. 244, 188-192, 1989; FEBS Lett. 268, 27-31, 1990] and the highly sensitive reverse PCR reaction we found that G alpha o, G alpha i-1, G alpha i-3 and G alpha s species are present in oocyte stage VI, G alpha o mRNA being the most abundant transcript. G alpha o mRNA was further quantitated through oogenesis, unfertilized eggs and early embryogenesis stages by a competitive PCR reaction using an 'in vitro' deleted G alpha o mRNA as the internal standard. Using this approach we found that Xenopus G alpha o mRNA levels were constant during oogenesis and unfertilized eggs at a concentration of 3.5 pg of mRNA/stage (5 x 10(5) molecules) and diminish gradually during early embryogenesis, reaching a level of 0.3 pg in the gastrula stage. These findings show that oocyte G alpha o, and perhaps the rest of the alpha subunits, are expressed as maternal mRNAs and could play an important role in signal transduction at the beginning of oocyte cell differentiation.

Expression and potential functions of G-protein alpha subunits in embryos of Xenopus laevis.

During early embryonic development, many inductive interactions between tissues depend on signal transduction processes. We began to test the possibility that G-proteins participate in the signal transduction pathways that mediate neural induction. The expression during Xenopus development of three G alpha subunits, G alpha 0, G alpha i-1 and G alpha s-1, was characterized. The three maternally expressed genes showed different expression patterns during early development. Whole-mount in situ hybridization revealed that all three genes were expressed almost exclusively in the gastrula ectoderm and predominantly in the neuroectoderm in the neurula embryo. In order to investigate the involvement of these proteins in neural induction, we overexpressed the G-protein alpha subunits by injecting the G alpha mRNAs into fertilized eggs. Overexpression of G alpha s-1 increased the ability of gastrula ectoderm to become induced to neural tissue approximately four-fold. Overexpression of G alpha 0 and G alpha i-1 had less pronounced effects on neural competence, and inhibition of the G alpha 0 and G alpha i-1 proteins by pertussis toxin did not change the neural competence of the exposed gastrula ectoderm. Overexpression of the G alpha 0 and G alpha i-1 genes did, however, inhibit the normal disappearance of the blastocoel during gastrulation, suggesting a role for these G-proteins in regulating this process. The data also suggest a specific role for the G alpha s subunit in mediating the initial phases of neural induction.

Differential stimulation of the GTPase activity of G-proteins by polylysine.

Polylysine, polyornithine and, to a lesser extent, polyarginine were found to stimulate the GTPase activity of the purified recombinant alpha subunit of the human G(i)-3 transducing protein alpha i-3. Optimal stimulation of 4- to 5-fold was obtained with polylysine concentrations between 1 and 20 microM, higher concentrations being inhibitory. Polylysine at similar concentrations stimulated by 50% the GTPase of transducin (GT), the vision transducing protein, but had only a very slight effect on the GTPase of the p21 product of the H-ras protooncogene. The stimulation of the alpha i-3 GTPase caused by polylysine was due to a reduction of the apparent Km for GTP from 3.8 to 1.3 microM. The stimulation by polylysine was observed at free Mg2+ concentrations below 1 microM. These results indicate that polylysine acts in a fashion similar to mastoparan and substance P in mimicking the action of an agonist-bound receptor on G-proteins.

Structure and function of G proteins.

G proteins are heterotrimeric proteins involved in the transduction of a variety of external signals in all eukaryotic organisms. This review analyzes the molecular aspects of G protein structure and function. The cloning of cDNAs coding for a great variety of G protein subunits has allowed us to deduce the primary and secondary structure of the subunits. Emphasis is given to the dissection of the molecular regions of the G alpha subunits implicated in the binding and hydrolysis of GTP and in the interaction with the receptor, with the effector and with the beta gamma dimer. The localization of these regions in a two-dimensional model of the G alpha subunit is attempted to provide a more comprehensive view of the structure and function of G proteins.

Polylysine activates membrane-bound adenylyl cyclase from Xenopus laevis oocytes through the Gs transducing protein.

1. The activity of the adenylyl cyclase found in the membranes of Xenopus laevis can be affected by polylysine and other polycations. 2. The activity of the catalytic subunit measured with forskolin is inhibited by polylysine and polyarginine at concentrations above 10 microM and by spermine above 3 mM. 3. The adenylyl cyclase activity stimulated by GTP-gamma-S or F- through the stimulatory G protein (Gs) can be increased by polylysine, polyornithine and spermine but not by polyarginine. 4. Polylysine stimulation of Gs dependent activity is due to the increase in the apparent affinity for GTP-gamma-S and to a lowering of the requirement for Mg2+ concentration.

Molecular cloning of a novel splice variant of the alpha subunit of the mammalian Go protein.

We screened a HIT (hamster insulin-secreting tumor) cell cDNA library constructed in lambda gt11 with a Go-specific oligonucleotide probe and isolated six recombinant phages. The inserts of these phages encoded two forms of alpha o, called here alpha o1 and alpha o2. The deduced amino acid sequence of alpha o1 is identical in all of its 354 amino acids to that reported previously for rat and bovine alpha o; that of alpha o2, also of 354 amino acids, is identical to alpha o1 up to and including amino acid 248 and differs thereafter in 26 amino acids. At the nucleotide level, alpha o1 and alpha o2 are identical up to and including the second base of the codon that specifies amino acid 243 and differs thereafter in 88 nucleotides of the remaining open reading frame and has no similarity to alpha o1 in its 3'-untranslated region. We propose that alpha o1 and alpha o2 result as a consequence of alternative splicing of a single alpha o transcript. Northern analysis with specifically designed oligonucleotides indicates that both forms of alpha o are expressed in normal tissues, e.g. brain. After in vitro transcription and translation, the peptides encoded in the alpha o1 and alpha o2 cDNAs could be ADP-ribosylated by pertussis toxin in the presence of added beta gamma dimers. The count of distinct G proteins keeps increasing.

Molecular cloning and sequence determination of four different cDNA species coding for alpha-subunits of G proteins from Xenopus laevis oocytes.

A cDNA library prepared from Xenopus laevis oocytes in lambda gt10 was screened with a mixture of three oligonucleotide probes designed to detect sequences found in different mammalian genes coding for alpha-subunits of G-proteins. In addition to a clone coding for a G alpha o-type subunit previously reported [(1989) FEBS Lett. 244, 188-192] four additional clones have been found coding for different G alpha protein subunits. By comparison with mammalian alpha-subunits, these oocyte cDNAs correspond to two closely related G alpha s-1a, to a G alpha i-1 and to a G alpha i-3 species. The derived amino acid sequences showed that both G alpha s species contain 379 residues, corresponding to the short species without the serine residue and with a calculated Mr of 42720. The G alpha i-1 gene encodes a 354 amino acid protein with an Mr of 39,000 and the G alpha i-3 encodes an incomplete open reading frame of 345 residues, lacking the first 9 amino acid residues at the NH2 terminus. All these G alpha-subunits showed high identity with their respective mammalian counterparts (75-80%), indicating a great degree of conservation through the evolution and the important cellular regulatory function that they play.

Molecular cloning and sequence determination of a cDNA coding for the alpha-subunit of a Go-type protein of Xenopus laevis oocytes.

Xenopus laevis oocytes are cells ideally suited to the study of signal transduction and of the G-proteins that are involved in this process. A X. laevis cDNA library in lambda gt10 has been screened with a mixture of three oligonucleotide probes designed to detect sequences found in various mammalian alpha-subunits of G-proteins. One of these clones has been purified through tertiary screening and the DNA insert has been sequenced. This clone was found to include the total sequence coding for a 354 amino acid protein that is 89% identical to the sequence of alpha-subunit of rat Go. The differences with the mammalian protein were clustered in amino acids 290-315, which have been postulated to define the region interacting with the receptor and effector molecule. The homology with the alpha-subunits of other mammalian G-proteins is lower (65-70% to Gi and 42% to Gs). On this basis, this clone can be classified as Go-like.