Clonal variation of adenylyl cyclase activity in a rat tumor cell line caused by change in G protein‐catalytic unit interaction

Two subclones of the rat XC cell line characterized by different morphology exhibited quite different adenylyl cyclase responses upon various stimulations. Upon treatment with cholera toxin, clone RK1 accumulated a high level of intracellular cAMP thereby changing its polygonal morphology to an elongated morphology, while the other clone, LK1, with a fibroblastic morphology, failed to increase the intracellular cAMP and remained morphologically unchanged. When membrane fractions derived from these two clones were stimulated with 10 μM forskolin, 10 μM GTPλS, or 10 mM NaF, five‐ to 20‐fold more cAMP was accumulated in RK1‐derived membranes than in LK1‐derived membranes. With the same membrane fractions, upon treatment with Mn++, which directly stimulates the catalytic unit, a high level of cAMP was accumulated both in RK1 and LK1, indicating that the catalytic function inducible by Mn++ was similar in both clones. There was no significant difference in the level of expression of G protein α2, αi (at least αi1 and αi2), and β subunits between LK1 and RK1. Cholateextracts of the membrane protein of LK1 and RK1 reconstituted the adenylyl cyclase activity of the cyc variant of S49 lymphoma cells to the same level. Therefore, it is inferred that the defect in LK1 resides in the interaction of stimulatory G proteins and the actual catalyst.

[1]  C. Slaughter,et al.  Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. , 1989, Science.

[2]  R. Toyama,et al.  Presence of three distinct molecular species of Gi protein alpha subunit. Structure of rat cDNAs and human genomic DNAs. , 1988, The Journal of biological chemistry.

[3]  David E. Clapham,et al.  Roles of G protein subunits in transmembrane signalling , 1988, Nature.

[4]  D. T. Jones,et al.  Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. , 1987, The Journal of biological chemistry.

[5]  A. Gilman,et al.  A second form of the beta subunit of signal-transducing G proteins. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[6]  T. Katada,et al.  A new GTP‐binding protein in brain tissues serving as the specific substrate of islet‐activating protein, pertussis toxin , 1987, FEBS letters.

[7]  H. Bourne,et al.  Inhibitory and stimulatory G proteins of adenylate cyclase: cDNA and amino acid sequences of the alpha chains. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[8]  A. Gilman,et al.  Molecular basis for two forms of the G protein that stimulates adenylate cyclase. , 1986, The Journal of biological chemistry.

[9]  H. Kawasaki,et al.  Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Brian K. Kobilka,et al.  Cloning of the gene and cDNA for mammalian β-adrenergic receptor and homology with rhodopsin , 1986, Nature.

[11]  K. Kangawa,et al.  Primary structure of the α‐subunit of bovine adenylate cyclase‐inhibiting G‐protein deduced from the cDNA sequence , 1986, FEBS letters.

[12]  T. Hirose,et al.  Primary structure of the α‐subunit of bovine adenylate cyclase‐stimulating G‐protein deduced from the cDNA sequence , 1986, FEBS letters.

[13]  G. Strewler,et al.  Activation of the parathyroid hormone receptor-adenylate cyclase system in osteosarcoma cells by a human renal carcinoma factor. , 1985, Cancer research.

[14]  K. Sugimoto,et al.  Primary structure of the β‐subunit of bovine transducin deduced from the cDNA sequence , 1985, FEBS letters.

[15]  M. Simon,et al.  Isolation and characterization of a cDNA clone for the gamma subunit of bovine retinal transducin. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R. Iyengar,et al.  Glucagon-induced heterologous desensitization of the MDCK cell adenylyl cyclase. Increases in the apparent levels of the inhibitory regulator (Ni). , 1984, The Journal of biological chemistry.

[17]  B. Schimmer,et al.  Isolation of forskolin-resistant adrenal cells defective in the adenylate cyclase system. , 1984, The Journal of biological chemistry.

[18]  A. Gilman.  G proteins and dual control of adenylate cyclase , 1984, Cell.

[19]  N. Shimada,et al.  GDP does not mediate but rather inhibits hormonal signal to adenylate cyclase. , 1983, The Journal of biological chemistry.

[20]  R. Butcher,et al.  Differences in the forskolin activation of adenylate cyclases in wild-type and variant lymphoma cells. , 1982, Molecular pharmacology.

[21]  H. Lester,et al.  ADP‐Ribosylation of Membrane Proteins in Cholinergic Nerve Terminals , 1982, Journal of neurochemistry.

[22]  J. Daly,et al.  Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. , 1981, The Journal of biological chemistry.

[23]  A. Gilman,et al.  Purification of the regulatory component of adenylate cyclase. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[24]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[25]  A. Gilman,et al.  Reconstitution of catecholamine-sensitive adenylate cyclase. Association of a regulatory component of the enzyme with membranes containing the catalytic protein and beta-adrenergic receptors. , 1979, The Journal of biological chemistry.

[26]  H. Yoshikura,et al.  Unstable resistance of G mouse fibroblasts to ecotropic murine leukemia virus infection , 1979, Journal of virology.

[27]  M. Artman,et al.  Nicotinamide adenine dinucleotide splitting enzyme: a characteristic of the mouse macrophage. , 1978, Science.

[28]  H. Bourne,et al.  Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase. , 1978, The Journal of biological chemistry.

[29]  A. Gilman,et al.  Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. , 1978, The Journal of biological chemistry.

[30]  D. Gill,et al.  ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[31]  A. Gilman,et al.  Reconstitution of catecholamine-sensitive adenylate cyclase activity: interactions of solubilized components with receptor-replete membranes. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[32]  M. Maguire,et al.  Relationship between the beta-adrenergic receptor and adenylate cyclase. , 1977, The Journal of biological chemistry.

[33]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[34]  P. Cuatrecasas,et al.  Mobility of cholera toxin receptors on rat lymphocyte membranes. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[35]  H. Bourne,et al.  Selection of a variant lymphoma cell deficient in adenylate cyclase. , 1975, Science.

[36]  A. Gilman,et al.  Cyclic Adenosine Monophosphate and Alteration of Chinese Hamster Ovary Cell Morphology: a Rapid, Sensitive In Vitro Assay for the Enterotoxins of Vibrio cholerae and Escherichia coli , 1974, Infection and immunity.

[37]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[38]  J. Chirgwin,et al.  Isolation of RNA using guanidinium salts. , 1987, Methods in enzymology.

[39]  Fukasawa Toshio,et al.  Expression of the human adenovirus E1a product in yeast. , 1987 .

[40]  A. Gilman,et al.  G proteins: transducers of receptor-generated signals. , 1987, Annual review of biochemistry.

[41]  L. Birnbaumer,et al.  ADP-ribosylation of membrane components by pertussis and cholera toxin. , 1985, Methods in enzymology.

[42]  H. Bourne,et al.  Novel S49 lymphoma variants with aberrant cyclic AMP metabolism. , 1981, Molecular pharmacology.

[43]  A. Gilman,et al.  Biochemical properties of hormone-sensitive adenylate cyclase. , 1980, Annual review of biochemistry.

[44]  O. Hayaishi,et al.  Poly(ADP-ribose) and ADP-ribosylation of proteins. , 1977, Annual review of biochemistry.