Type VIII adenylyl cyclase in rat beta cells: coincidence signal detector/generator for glucose and GLP-1.

AIMS/HYPOTHESIS: The secretory function of pancreatic beta cells is synergistically stimulated by two signalling pathways which mediate the effects of nutrients and hormones such as glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) or glucagon. These hormones are known to activate adenylyl cyclase in beta cells. We examined the type of adenylyl cyclase that is associated with this synergistic interaction. METHODS: Insulin release, cAMP production, adenylyl cyclase activity, mRNA and protein expression were measured in fluorescence-activated cell sorter-purified rat beta cells and in the rat beta-cell lines RINm5F, INS-1 832/13 and INS-1 832/2. RESULTS: In primary beta cells, glucagon and GLP-1 synergistically potentiate the stimulatory effect of 20 mmol/l glucose on insulin release and cAMP production. Both effects are abrogated in the presence of the L-type Ca(2+)-channel blocker verapamil. The cAMP-producing activity of adenylyl cyclase in membranes from RINm5F cells is synergistically increased by Ca(2+)-calmodulin and recombinant GTP(gamma)S-activated G(s alpha)-protein subunits. This type of regulation is characteristic for type I and type VIII AC isoforms. Consistent with this functional data, AC mRNA analysis shows abundant expression of type VI AC, four splice variants of type VIII AC and low expression level of type I AC in beta cells. Type VIII AC expression at the protein level was observed using immunoblots of RINm5F cell extracts. CONCLUSION/INTERPRETATION: This study identifies type VIII AC in insulin-secreting cells as one of the potential molecular targets for synergism between GLP-1 receptor mediated and glucose-mediated signalling.

Isozyme-dependent sensitivity of adenylyl cyclases to P-site-mediated inhibition by adenine nucleosides and nucleoside 3'-polyphosphates.

Recombinant adenylyl cyclase isozyme Types I, II, VI, VII, and three splice variants of Type VIII were compared for their sensitivity to P-site-mediated inhibition by several adenine nucleoside derivatives and by the family of recently synthesized adenine nucleoside 3'-polyphosphates (Désaubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 14028-14034). Inhibitory potencies were dependent on isozyme type, the mode of activation of the respective isozymes, and on P-site ligand. For the nucleoside derivatives potency typically followed the order 2',5'-dideoxyadenosine (2',5'-ddAdo) > beta-adenosine > 9-(cyclopentyl)-adenine (9-CP-Ade) >/= 9-(tetrahydrofuryl)-adenine (9-THF-Ade; SQ 22,536), with the exception of Type II adenylyl cyclase, which was essentially insensitive to inhibition by 9-CP-Ade. For the adenine nucleoside 3'-polyphosphates inhibitory potency followed the order Ado < 2'-dAdo < 2',5'-ddAdo and 3'-mono- < 3'-di- < 3'-triphosphate. Differences in potency of these ligands were noted between isozymes. The most potent ligand was 2',5'-dd-3'-ATP with IC50 values of 40-300 nM. The data demonstrate isozyme selectivity for some ligands, suggesting the possibility of isozyme-selective inhibitors to take advantage of differences in P-site domains among adenylyl cyclase isozymes. Differential expression of adenylyl cyclase isozymes may dictate the physiological sensitivity and hence importance of this regulatory mechanism in different cells or tissues.

Splice variants of type VIII adenylyl cyclase. Differences in glycosylation and regulation by Ca2+/calmodulin.

Three alternatively spliced type VIII adenylyl cyclase messages have been identified by cDNA cloning and amplification from rat brain cDNA. Type VIII-A was previously referred to simply as type VIII (Cali, J. J., Zwaagstra, J. C., Mons, N., Cooper, D. M. F., and Krupinski, J. (1994) J. Biol. Chem. 269, 12190-12195). The types VIII-B and -C cDNAs differ from that of type VIII-A by deletion of 90 and 198 base pair exons, respectively, which encode a 30-amino acid extracellular domain with two consensus sites for N-linked glycosylation and a 66-amino acid cytoplasmic domain. Stable expression of types VIII-A, -B, and -C cDNAs in human embryonal kidney 293 (HEK-293) cells leads to the appearance of novel proteins, which are recognized by type VIII-specific antibodies and which co-migrate with immunoreactive species detected on immunoblots of rat brain membranes. Types VIII-A and -C are modified by N-linked glycosylation, while type VIII-B is insensitive to treatment with N-glycosidase F. An influx of extracellular Ca2+ stimulates cAMP accumulation in HEK-293 cells stably expressing type VIII-A, -B, or -C, but not in control cells. Adenylyl cyclase activity of each of the variants is stimulated by Ca2+/calmodulin and the EC50 for activation of type VIII-C is one fourth of that for either type VIII-A or -B. Type VIII-C also has a distinct Km for substrate, which is approximately 4-12-fold higher than that for types VIII-A or -B depending on whether Mn2+ or Mg2+ is the counterion for ATP. The differences in the structural and enzymatic properties of these three variants are discussed.

Type VIII adenylyl cyclase. A Ca2+/calmodulin-stimulated enzyme expressed in discrete regions of rat brain.

A cDNA that encodes type VIII adenylyl cyclase has been isolated from two rat brain libraries. The open reading frame encodes a 1248-amino acid protein predicted to have two sets of six transmembrane spans and two putative nucleotide binding domains as is characteristic of other mammalian adenylyl cyclases. Two type VIII messages are detected in rat brain with estimated sizes of 5.5 and 4.4 kilobases. In situ hybridization indicates that the type VIII messages are most abundantly expressed in the granule cells of the dentate gyrus, the pyramidal cells of hippocampal fields CA1-CA3, the entorhinal cortex, and the piriform cortex. Hybridization is also detected in the neocortex, the amygdaloid complex, and regions of the thalamus and hypothalamus. Stable expression of the type VIII cDNA in human embryonal kidney cells leads to the appearance of a novel 165-kDa glycoprotein in the membrane fraction. Stimulation of these cells with agents that increase intracellular Ca2+ results in up to 43-fold increases in cAMP accumulation over that of control cells transfected with the expression vector. Addition of isoproterenol alone does not lead to type VIII-specific effects in intact cells. Adenylyl cyclase activity in membranes prepared from type VIII-transformed cells is stimulated up to 40-fold by the addition of Ca2+/calmodulin (EC50 = 53 nM calmodulin). The addition of activated recombinant alpha subunit of Gs synergistically increases the Ca2+/calmodulin-stimulated activity. A possible role for type VIII adenylyl cyclase in long-term potentiation is discussed.

Frameshift and splice-junction mutations in the sterol 27-hydroxylase gene cause cerebrotendinous xanthomatosis in Jews or Moroccan origin.

The sterol 27-hydroxylase (EC 1.14.13.15) catalyzes steps in the oxidation of sterol intermediates that form bile acids. Mutations in this gene give rise to the autosomal recessive disease cerebrotendinous xanthomatosis (CTX). CTX is characterized by tendon xanthomas, cataracts, a multitude of neurological manifestations, and premature atherosclerosis. A relatively high prevalence of the disease has been noted in Jews originating from Morocco. The major objectives of the present investigation were to determine the gene structure and characterize the common mutant alleles that cause CTX in Moroccan Jews. The gene contains nine exons and eight introns and encompasses at least 18.6 kb of DNA. The putative promoter region is rich in guanidine and cytosine residues and contains potential binding sites for the transcription factor Sp1 and the liver transcription factor, LF-B1. Blotting analysis revealed that the mutant alleles do not produce any detectable sterol 27-hydroxylase mRNA. No major gene rearrangements were found and single-strand conformational polymorphism followed by sequence analysis identified two underlying mutations: deletion of thymidine in exon 4 and a guanosine to adenosine substitution at the 3' splice acceptor site of intron 4 of the gene. The molecular characterization of CTX in Jews of Moroccan origin provides a definitive diagnosis of this treatable disease.

Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning.

The GTP-binding regulatory proteins (G proteins) that transduce signals from receptors to effectors are composed of alpha, beta, and gamma subunits. Whereas the role of alpha subunits in directly regulating effector activity is widely accepted, it has recently been demonstrated that beta gamma subunits may also directly regulate effector activity. This has made clear the importance of identifying and characterizing beta and gamma subunits. We have isolated a cDNA clone encoding a new gamma subunit, referred to here as the gamma 7 subunit, using probes based on peptide sequences of a gamma subunit previously purified from bovine brain. The clone contains a 1.47-kilobase cDNA insert, which includes an open reading frame of 204 base pairs that predicts a 68-amino acid polypeptide with a calculated M(r) of 7553. The predicted protein shares amino acid identities with the other known gamma subunits, ranging from 38 to 68%. Also characteristic of gamma subunits is a carboxyl-terminal CAAX motif. The expression of the gamma 7 subunit as well as the gamma 2, gamma 3, and gamma 5 subunits was examined in several bovine tissues at both the mRNA and protein levels. Whereas the gamma 2 and gamma 3 subunits were selectively expressed in brain, the gamma 5 and gamma 7 subunits were expressed in a variety of tissues. Thus, the gamma 5 and gamma 7 subunits are the first G protein gamma subunits known that could participate in the regulation of widely distributed signal transduction pathways.

Cloning of the human cholesterol 7 alpha-hydroxylase gene (CYP7) and localization to chromosome 8q11-q12.

Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is a microsomal cytochrome P450 that catalyzes the first step in bile acid synthesis. In this paper, we describe the cloning, characterization, and chromosomal mapping of the human 7 alpha-hydroxylase gene (CYP7). The gene spans 10 kb and contains six exons and five introns. The exon-intron boundaries are completely conserved between the human and rat genes. Sequencing of the 5' flanking region revealed consensus recognition sequences for a number of liver-specific transcription factors. The human CYP7 gene was mapped to chromosome 8q11-q12 using both mouse-human somatic cell hybrids and in situ chromosomal hybridization studies. A total of four single-stranded conformation-dependent DNA polymorphisms and an Alu sequence-related polymorphism were identified. Of the individuals analyzed, 80% were heterozygous for at least one of these five polymorphisms. The localization and characterization of the human 7 alpha-hydroxylase gene, as well as the identification of polymorphisms, provide the molecular tools necessary to investigate the role of this gene in disorders of cholesterol and bile acid metabolism.

Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis.

The sterol storage disorder cerebrotendinous xanthomatosis (CTX) is characterized by abnormal deposition of cholesterol and cholestanol in multiple tissues. Deposition in the central nervous system leads to neurological dysfunction marked by dementia, spinal cord paresis, and cerebellar ataxia. Deposition in other tissues causes tendon xanthomas, premature atherosclerosis, and cataracts. In two unrelated patients with CTX, we have identified different point mutations in the gene (CYP27) encoding sterol 27-hydroxylase, a key enzyme in the bile acid biosynthesis pathway. Transfection of mutant cDNAs into cultured cells results in the synthesis of immunoreactive sterol 27-hydroxylase protein with greatly diminished enzyme activity. We have localized the CYP27 gene to the q33-qter interval of human chromosome 2, and to mouse chromosome 1, in agreement with the autosomal recessive inheritance pattern of CTX. These findings underscore the essential role played by sterols in the central nervous system and suggest that mutations in other sterol metabolizing enzymes may contribute to diseases with neurological manifestations.

Characterization of human sterol 27-hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple oxidation reaction in bile acid biosynthesis.

The enzyme sterol 27-hydroxylase catalyzes the first step in the oxidation of the side chain of sterol intermediates in the bile acid synthesis pathway. Human sterol 27-hydroxylase cDNAs were isolated from a liver cDNA library by cross-hybridization with a previously cloned rabbit cDNA probe. DNA sequence analysis of hybridization-positive clones predicted a human sterol 27-hydroxylase consisting of a 33-amino-acid mitochondrial signal sequence followed by a mature protein of 498 amino acids. RNA blotting experiments demonstrated sterol 27-hydroxylase mRNAs of approximately 1.8 to 2.2 kilobases in liver and fibroblast cells. The steady state levels of the mRNA did not change when cultured cells were grown in the presence or absence of sterols. Introduction of the sterol 27-hydroxylase cDNA into Simian COS cells resulted in the expression of active enzyme capable of catalyzing multiple oxidation reactions (R-CH3----R-CH2OH----R-COOH) at carbon 27 of sterol intermediates of the bile acid synthesis pathway.