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Biochem J. Jan 1, 1997; 321(Pt 1): 177–185.
PMCID: PMC1218052

Intracellular localization of the PDE4A cAMP-specific phosphodiesterase splice variant RD1 (RNPDE4A1A) in stably transfected human thyroid carcinoma FTC cell lines.


Cells of two human follicular thyroid carcinoma cell lines (FTC133, FTC236) were stably transfected with a cDNA encoding the PDE4A cAMP-specific phosphodiesterase (PDE) splice variant RD1 (RNPDE4A1A) so as to generate the cloned cell lines, FTC133A and FTC236A. This allowed the expression of a novel rolipram-inhibited cAMP-specific PDE activity in these cells. Unlike the parent cell lines in which Ca2+/calmodulin caused a profound activation (approx. 3-4-fold) of homogenate PDE activity, no such stimulation was evident in the RD1-expressing cell lines, indicating loss of PDE1 activity. Reverse transcriptase-PCR analysis indicated that this was due to the down-regulation of the PDE1C isoform. The novel PDE4 activity in transfected cells was located exclusively in the membrane fraction, as was immunoreactive RD1. Low concentrations of the detergent Triton X-100, but not high NaCl concentrations, allowed RD1 to be solubilized. Laser scanning confocal immunofluorescence analyses identified RD1 immunoreactivity in a discrete perinuclear region of these RD1-expressing transfected cell lines. A similar pattern of labelling was observed using the antiserum Tex1, which specifically identified the Golgi apparatus. Treatment of FTC133A cells with the Golgi-perturbing agents monensin and brefeldin A led to a similar redistribution of immunoreactive species detected using both the Tex1 and anti-RD1 antisera. It is suggested that the PDE4A splice variant RD1 contains a membrane-association signal which allows the targeted expression of RD1 within the Golgi complex of these human follicular thyroid carcinoma cell lines.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Beavo JA. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev. 1995 Oct;75(4):725–748. [PubMed]
  • Bolger GB. Molecular biology of the cyclic AMP-specific cyclic nucleotide phosphodiesterases: a diverse family of regulatory enzymes. Cell Signal. 1994 Nov;6(8):851–859. [PubMed]
  • Conti M, Nemoz G, Sette C, Vicini E. Recent progress in understanding the hormonal regulation of phosphodiesterases. Endocr Rev. 1995 Jun;16(3):370–389. [PubMed]
  • Manganiello VC, Murata T, Taira M, Belfrage P, Degerman E. Diversity in cyclic nucleotide phosphodiesterase isoenzyme families. Arch Biochem Biophys. 1995 Sep 10;322(1):1–13. [PubMed]
  • Torphy TJ, DeWolf WE, Jr, Green DW, Livi GP. Biochemical characteristics and cellular regulation of phosphodiesterase IV. Agents Actions Suppl. 1993;43:51–71. [PubMed]
  • Davis RL, Takayasu H, Eberwine M, Myres J. Cloning and characterization of mammalian homologs of the Drosophila dunce+ gene. Proc Natl Acad Sci U S A. 1989 May;86(10):3604–3608. [PMC free article] [PubMed]
  • Milatovich A, Bolger G, Michaeli T, Francke U. Chromosome localizations of genes for five cAMP-specific phosphodiesterases in man and mouse. Somat Cell Mol Genet. 1994 Mar;20(2):75–86. [PubMed]
  • Horton YM, Sullivan M, Houslay MD. Molecular cloning of a novel splice variant of human type IVA (PDE-IVA) cyclic AMP phosphodiesterase and localization of the gene to the p13.2-q12 region of human chromosome 19 [corrected]. Biochem J. 1995 Jun 1;308(Pt 2):683–691. [PMC free article] [PubMed]
  • Szpirer C, Szpirer J, Rivière M, Swinnen J, Vicini E, Conti M. Chromosomal localization of the human and rat genes (PDE4D and PDE4B) encoding the cAMP-specific phosphodiesterases 3 and 4. Cytogenet Cell Genet. 1995;69(1-2):11–14. [PubMed]
  • Sette C, Vicini E, Conti M. The ratPDE3/IVd phosphodiesterase gene codes for multiple proteins differentially activated by cAMP-dependent protein kinase. J Biol Chem. 1994 Jul 15;269(28):18271–18274. [PubMed]
  • Bolger GB, McPhee I, Houslay MD. Alternative splicing of cAMP-specific phosphodiesterase mRNA transcripts. Characterization of a novel tissue-specific isoform, RNPDE4A8. J Biol Chem. 1996 Jan 12;271(2):1065–1071. [PubMed]
  • McPhee I, Pooley L, Lobban M, Bolger G, Houslay MD. Identification, characterization and regional distribution in brain of RPDE-6 (RNPDE4A5), a novel splice variant of the PDE4A cyclic AMP phosphodiesterase family. Biochem J. 1995 Sep 15;310(Pt 3):965–974. [PMC free article] [PubMed]
  • Shakur Y, Wilson M, Pooley L, Lobban M, Griffiths SL, Campbell AM, Beattie J, Daly C, Houslay MD. Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum. Biochem J. 1995 Mar 15;306(Pt 3):801–809. [PMC free article] [PubMed]
  • Shakur Y, Pryde JG, Houslay MD. Engineered deletion of the unique N-terminal domain of the cyclic AMP-specific phosphodiesterase RD1 prevents plasma membrane association and the attainment of enhanced thermostability without altering its sensitivity to inhibition by rolipram. Biochem J. 1993 Jun 15;292(Pt 3):677–686. [PMC free article] [PubMed]
  • Scotland G, Houslay MD. Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase. Biochem J. 1995 Jun 1;308(Pt 2):673–681. [PMC free article] [PubMed]
  • Scott JD, McCartney S. Localization of A-kinase through anchoring proteins. Mol Endocrinol. 1994 Jan;8(1):5–11. [PubMed]
  • Smith KJ, Scotland G, Beattie J, Trayer IP, Houslay MD. Determination of the structure of the N-terminal splice region of the cyclic AMP-specific phosphodiesterase RD1 (RNPDE4A1) by 1H NMR and identification of the membrane association domain using chimeric constructs. J Biol Chem. 1996 Jul 12;271(28):16703–16711. [PubMed]
  • O'Connell JC, McCallum JF, McPhee I, Wakefield J, Houslay ES, Wishart W, Bolger G, Frame M, Houslay MD. The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5). Biochem J. 1996 Aug 15;318(Pt 1):255–261. [PMC free article] [PubMed]
  • Hoelting T, Zielke A, Siperstein AE, Clark OH, Duh QY. Aberrations of growth factor control in metastatic follicular thyroid cancer in vitro. Clin Exp Metastasis. 1994 Jul;12(4):315–323. [PubMed]
  • Hölting T, Siperstein AE, Clark OH, Duh QY. Epidermal growth factor (EGF)- and transforming growth factor alpha-stimulated invasion and growth of follicular thyroid cancer cells can be blocked by antagonism to the EGF receptor and tyrosine kinase in vitro. Eur J Endocrinol. 1995 Feb;132(2):229–235. [PubMed]
  • Korman AJ, Frantz JD, Strominger JL, Mulligan RC. Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2150–2154. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Spence S, Rena G, Sweeney G, Houslay MD. Induction of Ca2+/calmodulin-stimulated cyclic AMP phosphodiesterase (PDE1) activity in Chinese hamster ovary cells (CHO) by phorbol 12-myristate 13-acetate and by the selective overexpression of protein kinase C isoforms. Biochem J. 1995 Sep 15;310(Pt 3):975–982. [PMC free article] [PubMed]
  • Loughney K, Martins TJ, Harris EA, Sadhu K, Hicks JB, Sonnenburg WK, Beavo JA, Ferguson K. Isolation and characterization of cDNAs corresponding to two human calcium, calmodulin-regulated, 3',5'-cyclic nucleotide phosphodiesterases. J Biol Chem. 1996 Jan 12;271(2):796–806. [PubMed]
  • Yan C, Bentley JK, Sonnenburg WK, Beavo JA. Differential expression of the 61 kDa and 63 kDa calmodulin-dependent phosphodiesterases in the mouse brain. J Neurosci. 1994 Mar;14(3 Pt 1):973–984. [PubMed]
  • Yan C, Zhao AZ, Bentley JK, Loughney K, Ferguson K, Beavo JA. Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9677–9681. [PMC free article] [PubMed]
  • Nakamura N, Rabouille C, Watson R, Nilsson T, Hui N, Slusarewicz P, Kreis TE, Warren G. Characterization of a cis-Golgi matrix protein, GM130. J Cell Biol. 1995 Dec;131(6 Pt 2):1715–1726. [PMC free article] [PubMed]
  • Thompson WJ, Appleman MM. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry. 1971 Jan 19;10(2):311–316. [PubMed]
  • Marchmont RJ, Houslay MD. A peripheral and an intrinsic enzyme constitute the cyclic AMP phosphodiesterase activity of rat liver plasma membranes. Biochem J. 1980 May 1;187(2):381–392. [PMC free article] [PubMed]
  • Lavan BE, Lakey T, Houslay MD. Resolution of soluble cyclic nucleotide phosphodiesterase isoenzymes, from liver and hepatocytes, identifies a novel IBMX-insensitive form. Biochem Pharmacol. 1989 Nov 15;38(22):4123–4136. [PubMed]
  • Michie AM, Lobban M, Müller T, Harnett MM, Houslay MD. Rapid regulation of PDE-2 and PDE-4 cyclic AMP phosphodiesterase activity following ligation of the T cell antigen receptor on thymocytes: analysis using the selective inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and rolipram. Cell Signal. 1996 Feb;8(2):97–110. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Erdogan S, Houslay MD. Challenge of human Jurkat T-cells with the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant. Biochem J. 1997 Jan 1;321(Pt 1):165–175. [PMC free article] [PubMed]
  • Torphy TJ, Zhou HL, Foley JJ, Sarau HM, Manning CD, Barnette MS. Salbutamol up-regulates PDE4 activity and induces a heterologous desensitization of U937 cells to prostaglandin E2. Implications for the therapeutic use of beta-adrenoceptor agonists. J Biol Chem. 1995 Oct 6;270(40):23598–23604. [PubMed]
  • Verghese MW, McConnell RT, Lenhard JM, Hamacher L, Jin SL. Regulation of distinct cyclic AMP-specific phosphodiesterase (phosphodiesterase type 4) isozymes in human monocytic cells. Mol Pharmacol. 1995 Jun;47(6):1164–1171. [PubMed]
  • Swinnen JV, Joseph DR, Conti M. The mRNA encoding a high-affinity cAMP phosphodiesterase is regulated by hormones and cAMP. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8197–8201. [PMC free article] [PubMed]
  • Nicholson CD, Challiss RA, Shahid M. Differential modulation of tissue function and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes. Trends Pharmacol Sci. 1991 Jan;12(1):19–27. [PubMed]
  • Nicholson CD, Shahid M. Inhibitors of cyclic nucleotide phosphodiesterase isoenzymes--their potential utility in the therapy of asthma. Pulm Pharmacol. 1994 Feb;7(1):1–17. [PubMed]
  • Raeburn D, Souness JE, Tomkinson A, Karlsson JA. Isozyme-selective cyclic nucleotide phosphodiesterase inhibitors: biochemistry, pharmacology and therapeutic potential in asthma. Prog Drug Res. 1993;40:9–32. [PubMed]
  • Ashton MJ, Cook DC, Fenton G, Karlsson JA, Palfreyman MN, Raeburn D, Ratcliffe AJ, Souness JE, Thurairatnam S, Vicker N. Selective type IV phosphodiesterase inhibitors as antiasthmatic agents. The syntheses and biological activities of 3-(cyclopentyloxy)-4-methoxybenzamides and analogues. J Med Chem. 1994 May 27;37(11):1696–1703. [PubMed]
  • Sullivan M, Egerton M, Shakur Y, Marquardsen A, Houslay MD. Molecular cloning and expression, in both COS-1 cells and S. cerevisiae, of a human cytosolic type-IVA, cyclic AMP specific phosphodiesterase (hPDE-IVA-h6.1). Cell Signal. 1994 Sep;6(7):793–812. [PubMed]
  • Wilson M, Sullivan M, Brown N, Houslay MD. Purification, characterization and analysis of rolipram inhibition of a human type-IVA cyclic AMP-specific phosphodiesterase expressed in yeast. Biochem J. 1994 Dec 1;304(Pt 2):407–415. [PMC free article] [PubMed]
  • Conti M, Iona S, Cuomo M, Swinnen JV, Odeh J, Svoboda ME. Characterization of a hormone-inducible, high affinity adenosine 3'-5'-cyclic monophosphate phosphodiesterase from the rat Sertoli cell. Biochemistry. 1995 Jun 27;34(25):7979–7987. [PubMed]
  • Sonnenburg WK, Seger D, Beavo JA. Molecular cloning of a cDNA encoding the "61-kDa" calmodulin-stimulated cyclic nucleotide phosphodiesterase. Tissue-specific expression of structurally related isoforms. J Biol Chem. 1993 Jan 5;268(1):645–652. [PubMed]
  • Spence S, Houslay MD. The local anaesthetic benzyl alcohol attenuates the alpha 2-adrenoceptor-mediated inhibition of human platelet adenylate cyclase activity when stimulated by prostaglandin E1, but not that stimulated by forskolin. Biochem J. 1989 Dec 1;264(2):483–488. [PMC free article] [PubMed]
  • Podzuweit T, Nennstiel P, Müller A. Isozyme selective inhibition of cGMP-stimulated cyclic nucleotide phosphodiesterases by erythro-9-(2-hydroxy-3-nonyl) adenine. Cell Signal. 1995 Sep;7(7):733–738. [PubMed]
  • Méry PF, Pavoine C, Pecker F, Fischmeister R. Erythro-9-(2-hydroxy-3-nonyl)adenine inhibits cyclic GMP-stimulated phosphodiesterase in isolated cardiac myocytes. Mol Pharmacol. 1995 Jul;48(1):121–130. [PubMed]
  • Torii S, Banno T, Watanabe T, Ikehara Y, Murakami K, Nakayama K. Cytotoxicity of brefeldin A correlates with its inhibitory effect on membrane binding of COP coat proteins. J Biol Chem. 1995 May 12;270(19):11574–11580. [PubMed]
  • Qiu Z, Tufaro F, Gillam S. Brefeldin A and monensin arrest cell surface expression of membrane glycoproteins and release of rubella virus. J Gen Virol. 1995 Apr;76(Pt 4):855–863. [PubMed]
  • Pelham HR. Multiple targets for brefeldin A. Cell. 1991 Nov 1;67(3):449–451. [PubMed]
  • Klausner RD, Donaldson JG, Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol. 1992 Mar;116(5):1071–1080. [PMC free article] [PubMed]
  • Orci L, Tagaya M, Amherdt M, Perrelet A, Donaldson JG, Lippincott-Schwartz J, Klausner RD, Rothman JE. Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae. Cell. 1991 Mar 22;64(6):1183–1195. [PubMed]

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