Juan Olate

Oocyte adenylyl cyclase contains Ni, yet the guanine nucleotide-dependent inhibition by progesterone is not sensitive to pertussis toxin

The possible susceptibility to pertussis toxin of the guanine nucleotide‐dependent inhibition of oocyte adenylyl cyclase by progesterone was investigated. This action of progesterone is mediated by a membrane bound receptor as opposed to a receptor of cytosolic or nuclear localization. However, the inhibitory effect of progesterone was unaffected by pertussis toxin, even though the oocyte membrane Ni was fully ADP‐ribosylated with pertussis toxin, as revealed by lack of further [32P]ADP‐ribosylation on subsequent re‐incubation with pertussis toxin. These results indicate that the action of progesterone, in spite of being nucleotide‐dependent, is either not mediated by Ni, suggesting the existence of an additional nucleotide regulatory component, or if mediated by Ni, involves a mode of regulation of this coupling protein that is different from that by which all other inhibitory hormones act on adenylyl cyclase.

Molecular cloning and sequence determination of a cDNA coding for the α-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 λgt10 has been screened with a mixture of three oligonucleotide probes designed to detect sequences found in various mammalian α‐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 α‐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 α‐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.

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

A cDNA library preprared from Xenopus laevis oocytes in λgt10 was screened with a mixture of three oligonucleotide probes designed to detect sequences found in different mammalian genes coding for a‐subunits of G‐proteins. In addition to a clone coding for a Gαo‐type subunit previously reported [(1989) FEBS Lett. 244, 188‐192] four additional clones have been found coding for different Gα protein subunits. By comparison with mammalian α‐subunits, these oocyte cDNAs correspond to two closely related Gas‐la, to a Gαi‐1 and to a Gαi‐3 species. The derived amino acid sequences showed that both Gαs species contain 379 residues, corresponding to the short species without the serine residue and with a calculated M r of 42720. The Gαi‐1 gene encodes a 354 amino acid protein with an M r, of 39000 and the Gαi‐3 encodes an incomplete open reading frame of 345 residues, lacking the first 9 amino acid residues at the NH2, terminus. All these Gα‐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.

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.

Human-Xenopus chimeras of Gsα 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 α 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 Gsα subunits are 92% identical to those found in the short form of human Gsα. Despite this high homology, the X. laevis Gsα 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 Gsα 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 Gsα. 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 Gsα. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTPγs and isoproterenol. The results obtained here clearly indicate that the Gα sequence that extends from amino acid 70 to 140, is important for the functionality of human Gsα in activating adenylyl cyclase.

Molecular basis of regulation of ionic channels by G proteins

Publisher Summary This chapter presents primary structure of G proteins as deduced from purified proteins and cloned subunits. It also discusses their functions and presents data on direct regulation of ionic channels by G proteins. Experiments on expression of subunits, either in bacteria or by in vitro translation of messenger RNA (mRNA) synthesized from complimentary DNA (cDNA), are discussed as tools for definitive assignment of function to a given G protein. The chapter also discusses the dynamics of G protein-mediated signal transduction. The key points discussed in the chapter include the existence of two superimposed regulatory cycles in which G proteins dissociate into α plus βγ upon activation by guanosine triphosphate (GTP) and where the dissociated α subunits hydrolyze GTP. The chapter emphasizes the action of receptors to catalyze rather than regulate by allostery the activation of G proteins by GTP and also the role of subunit dissociation, without which receptors cannot act as catalysts. It also provides an overview on intramembrane networking of G protein-mediated receptor-effector coupling.

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

The expression of mRNAs coding for different Xenopus laevis oocyte Gα subunits was analyzed by the PCR technique. Using the nucleotide sequences of five previously cloned cDNAs for oocyte Gα subunits [FEBS Lett. 244, 188–192, 1989; FEBS Lett. 268, 27–31, 1990] and the highly sensitive reverse PCR reaction we found that Gαo, Gαi‐1, Gαi‐3 and Gαs species are present in oocyte stage VI, Gαo mRNA being the most abundant transcript. Gαo mRNA was further quantitated through oogenesis, unfertilized eggs and early embryogenesis stages by a competitive PCR reaction using an ‘in vitro’ deleted Gαo mRNA as the internal standard. Using this approach we found that Xenopus Gαo mRNA levels were constant during oogenesis and unfertilized eggs at a concentration of 3.5 pg of mRNA/stage (5 × 105 molecules) and diminish gradually during early embryogenesis, reaching a level of 0.3 pg in the gastrula stage. These findings show that oocyte Gαo, and perhaps the rest of the α subunits, are expressed as maternal mRNAs and could play an important role in signal transduction at the beginning of oocyte cell differentiation.

Alterations in mouse liver monooxygenases by benzothiadiazoles

Abstract Administration of 1,2,3-benzothiadiazoles to mice had a biphasic effect on liver microsomal monooxygenases. During the first 15 hr of treatment, an inhibition of the in vivo metabolism of hexobarbital, as well as of the in vitro hydroxylation of naphthalene and N -demethylation of aminopyrine, was observed. An apparent decrease in cytochrome P-450 and in the activity of the NADPH-cytochrome c reductase also occurred. The levels of cytochrome b 5 and NADH-cytochrome c reductase activity were only slightly affected. A shift to 452 nm in the carbon monoxide difference spectrum was obtained with dithionite-reduced microsomes and this was not modified by ferricyanide. After the initial inhibitory phase, an enhancement of drug-metabolizing activities in vivo and in vitro and in the levels of some components of the mixed function oxidase system was observed. The carbon monoxide difference spectra of dithionite-reduced microsomes returned to a maximal absorption at 450 nm. The stimulatory effect on monooxygenase activity, elicited by benzothiadiazoles, was prevented completely by actinomycin D and was accompanied by increases in liver weight, microsomal protein, and incorporation of labeled amino acids into microsomal protein, as well as by proliferation of smooth and rough endoplasmic reticulum. Acrylamide gel analysis of liver microsomes from mice, given a single dose of 6-chloro-1, 2,3-benzothiadiazole 48 hr prior to being killed, showed preferential induction of cytochrome P-450 apoproteins of 50,000, 52,000 and 53,000 molecular weight.

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.

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.