Demonstration of the presence of G-proteins in hepatic microsomal fraction

The presence of G-proteins in isolated hepatic microsomal vesicles is demonstrated. The G-proteins were identified by their capacity to be ADP-ribosylated by cholera and pertussis toxins. Cholera toxin identified 42 and 45 kDa proteins, corresponding to alpha s-1 and alpha s-2, respectively. Pertussis toxin identified a 40 kDa protein corresponding to alpha i. The microsomal G-proteins are identical to the corresponding G proteins of the plasma membrane, but are present in different proportions; the microsomes have considerably less alpha s proteins than the plasma membrane.     

GTP hydrolysis by pure Ni, the inhibitory regulatory component of adenylyl cyclases

The stimulatory and inhibitory regulatory components of adenylyl cyclase (Ns and Ni), purified to apparent homogeneity without the use of regulatory ligands such as Mg, NaF, and guanyl-5'-yl imidodiphosphate, were tested for GTPase activity by incubating them with [gamma-32P]GTP and measuring 32Pi liberation using a charcoal adsorption assay to separate hydrolyzed from nonhydrolyzed radioactivity. We found that Ni is capable of hydrolyzing GTP. The activity was shown to be due to Ni itself and not to presence of one of its minor contaminants by correlating activity with abundance of the 40,000 Da alpha i subunit throughout the last stages of purification and by showing co-migration on a sucrose density gradient of the GTP-hydrolyzing activity with the alpha i, beta, and gamma subunits of Ni and not with any one of three minor contaminants present in the preparation tested. Preparations of Ns, free of detectable Ni, exhibited less than 10% the capacity to hydrolyze GTP, as compared to Ni on an equal protein basis. The basic properties of the GTP-hydrolyzing activity of Ni were determined. The activity is dependent on Mg ion (apparent Km = 5 to 15 nM), and is rapidly lost upon incubation with Mg2+ in the absence of GTP. MgGTP and free GTP serve equally well as substrate (apparent Km about 40 nM). Isotopic dilution studies indicate that the GTP binding site has a relative affinity for guanine nucleotides in the order GTP = GTP gamma S greater than GDP = GMP-P(NH)P greater than GDP beta S with the highest difference (GTP versus GDP beta S) being about 10-fold. NaF inhibited GTP hydrolysis by Ni at concentrations at which it activates Ni in intact membranes.     

Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes

Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification by molecular cloning of two forms of the alpha-subunit of the human liver stimulatory (GS) regulatory component of adenylyl cyclase

Two DNA molecules complementary to human liver mRNA coding for the alpha-subunit of the stimulatory regulatory component Gs of adenylyl cyclase were cloned. One of the two forms is a full-length cDNA of 1614 nucleotides plus a poly(A) tail of 59 nucleotides. The deduced sequence of 394 amino acids encoded by its open reading frame is essentially identical to that of the alpha-subunits of Gs identified by molecular cloning from bovine adrenals, bovine brain and rat brain. Two independent clones of the other type of cDNA were isolated. Both were incomplete, beginning within the open reading frame coding for the alpha s polypeptide. One codes for amino acids 5 through 394 and the other for amino acids 48 through 394 of the above described cDNA of 1614 nucleotides, and both have the identical 3'-untranslated sequence. They differ from the first cDNA, however, in that they lack a stretch of 42 nucleotides (numbers 214 through 255) and have nucleotides 213 (G) and 256 (G) replaced with C and A, respectively. This results in a predicted amino acid composition of another alpha-subunit of Gs that is shorter by 14 amino acids and contains two substitutions (Asp for Glu and Ser for Gly) at the interface between the deletion and the unchanged sequence. We call the smaller subunit alpha s1 and the larger alpha s2. This is the first demonstration of a structural heterogeneity in alpha s subunits that is due to a difference in amino acid sequence.     

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Mesodern-inducing factors and mesodermal patterning

Identification of the signalling molecules involved in mesoderm formation in amphibian embryos still presents problems. None of the original candidates, such as activin, have been definitively ruled out, and the new factors, such as the nodal-related genes, have come on to the scene. Of the original candidates, activin has been definitively shown to act as a morphogen, whereas bone morphogenetic protein (BMP)-4 has emerged as a ventral inducer and an inhibitor of neural differentiation. The effects of BMP-4 are antagonized by chordin, a molecule related to the product of the Drosophila gene short gastrulation.     

Gamma-subunits of G proteins, but not their alpha- or beta-subunits, are polyisoprenylated. Studies on post-translational modifications using in vitro translation with rabbit reticulocyte lysates

Lipid modifications that may be introduced into several subunits of G proteins were explored by in vitro translation of recombinant mRNAs in reticulocyte lysates. In agreement with studies by others, myristic acid was incorporated into alpha i's and alpha o, but not alpha s, beta, or gamma's. In contrast, mevalonate (Mev) was incorporated only into gamma-subunits. Both, the gamma-subunit of transducin (gamma T) and that of other G proteins (gamma G) were modified by the lysates but with different characteristics. Labeled gamma T was unstable and was rapidly proteolyzed. Labeled gamma G was stable. The Mev-derivative in gamma G was sensitive to methyliodide and, after cleavage and chromatographic analysis, comigrated with the C20 polyisoprenol geranylgeraniol. This indicated that gamma G had been geranylgeranylated and that this polyisoprenoid was attached to the protein through a thioether linkage. It is thought that polyisoprenylation is defined by the COOH-terminal sequence Cys-A-A-X, where A is an aliphatic acid and X is any amino acid. Replacement by mutation of the Cys of the COOH-terminal -Cys-Ala-Ile-Leu sequence of gamma G with Ser abolished Mev incorporation, suggesting this Cys as the site of attachment of the geranylgeranyl moiety. Yet, Mev incorporation was less than 10% as much into gamma G with the Cys-A-A-X sequence -Cys-Ala-Ile-Trp. Consistent with geranylgeranylation, the C15 farnesyl moiety of farnesyl pyrophosphate was not incorporated into gamma G unless the incubations were fortified with Mev. In contrast, the farnesyl moiety was incorporated in an Mev-independent manner into gamma T (COOH terminus: -Cys-Val-Ile-Ser) and c-Ha-ras (COOH terminus: -Cys-Val-Leu-Ser) which are both farnesylated rather than geranylgeranylated. Thus, 1) separate enzymes appear to be involved in transferring farnesyl and geranylgeranyl groups to proteins, 2) structural factors other than the CAAX box contribute to the activity of the polyisoprenylating enzymes, and 3) this type of lipidation may be part of a proteolytic signaling system. Polyisoprenylation, which increases hydrophobicity of the derivatized protein, may play a role in anchoring not only ras but also G proteins to membranes.     

Ant genomics sheds light on the molecular regulation of social organization

Ants are powerful model systems for the study of cooperation and sociality. In this review, we discuss how recent advances in ant genomics have contributed to our understanding of the evolution and organization of insect societies at the molecular level.