The Calmodulin-dependent Phosphodiesterase Gene PDE1C Encodes Several Functionally Different Splice Variants in a Tissue-specific Manner
1996; Elsevier BV; Volume: 271; Issue: 41 Linguagem: Inglês
10.1074/jbc.271.41.25699
ISSN1083-351X
AutoresChen Yan, Allan Z. Zhao, J. Kelley Bentley, Joseph A. Beavo,
Tópico(s)Receptor Mechanisms and Signaling
ResumoWe report here the identification of cDNAs for three new mouse PDE1C splice variants and the characterization of their kinetics, regulation by Ca2+, sensitivities to inhibitors, and tissue/cellular expression patterns. Sequence analysis indicated that these three cDNAs (PDE1C1, PDE1C4, and PDE1C5), together with our previously reported PDE1C2 and PDE1C3, are alternative splice products of the PDE1C gene. The results from RNase protection analysis and in situ hybridization indicated that the expression of the different PDE1C splice variants is differentially regulated in a tissue/cell-specific manner. Particularly, high levels of PDE1C mRNAs were found in the olfactory epithelium, testis, and several regions of mouse brain such as cerebellar granule cells. All of these splice variants have similar kinetic properties, showing high affinities and approximately the same relative Vmax values for both cAMP and cGMP. However, they responded to Ca2+ stimulation differently. In addition, they show different sensitivities to the calmodulin-dependent phosphodiesterase inhibitors, KS505a and SCH51866. Substrate competition experiments suggested the presence of only one catalytic site on these PDE1C isozymes for both cAMP and cGMP. In summary, these findings suggest that the PDE1C gene undergoes tissue-specific alternative splicing that generates structurally and functionally diverse gene products. We report here the identification of cDNAs for three new mouse PDE1C splice variants and the characterization of their kinetics, regulation by Ca2+, sensitivities to inhibitors, and tissue/cellular expression patterns. Sequence analysis indicated that these three cDNAs (PDE1C1, PDE1C4, and PDE1C5), together with our previously reported PDE1C2 and PDE1C3, are alternative splice products of the PDE1C gene. The results from RNase protection analysis and in situ hybridization indicated that the expression of the different PDE1C splice variants is differentially regulated in a tissue/cell-specific manner. Particularly, high levels of PDE1C mRNAs were found in the olfactory epithelium, testis, and several regions of mouse brain such as cerebellar granule cells. All of these splice variants have similar kinetic properties, showing high affinities and approximately the same relative Vmax values for both cAMP and cGMP. However, they responded to Ca2+ stimulation differently. In addition, they show different sensitivities to the calmodulin-dependent phosphodiesterase inhibitors, KS505a and SCH51866. Substrate competition experiments suggested the presence of only one catalytic site on these PDE1C isozymes for both cAMP and cGMP. In summary, these findings suggest that the PDE1C gene undergoes tissue-specific alternative splicing that generates structurally and functionally diverse gene products. INTRODUCTIONHormones and neurotransmitters control the levels of the intracellular second messengers, cAMP and cGMP, by regulating the activity of cyclases and phosphodiesterases (PDEs). 1The abbreviations used are: PDEphosphodiesteraseCaM-PDEcalmodulin-dependent phosphodiesteraseORFopen reading frameKmMichaelis constantUTRuntranslated regionPCRpolymerase chain reactionbpbase pair(s)MOPS4-morpholinepropanesulfonic acid. At least seven different families of PDE are currently recognized (1Beavo J.A. Conti M. Heaslip R.J. Mol. Pharmacol. 1994; 46: 399-405PubMed Google Scholar, 2Beavo J.A. Physiol. Rev. 1995; 75: 725-748Crossref PubMed Scopus (1634) Google Scholar). Most families contain several distinct genes, and many of these genes encode multiple alternative splice variants in a tissue-specific manner. The facts that different PDEs have unique sequences in their catalytic and/or regulatory domains and that they are often selectively expressed in a limited number of cell types allow cell-specific regulation of cyclic nucleotide level by the PDEs. They also provides a basis for selective therapeutic intervention.The Ca2+/calmodulin-dependent PDEs (CaM-PDEs) compose one of the best known of the multiple PDE families. All CaM-PDEs are activated by calmodulin in the presence of calcium. It is thought that CaM-PDEs act as mediators between the Ca2+ and cyclic nucleotide second messenger systems that allow cyclic nucleotide-dependent processes to be regulated by increases in intracellular Ca2+ concentration (2Beavo J.A. Physiol. Rev. 1995; 75: 725-748Crossref PubMed Scopus (1634) Google Scholar). A rather large family of CaM-PDE isozymes is expressed in mammals. At least six different members, including 59-, 61-, 63-, 68-, and 75-kDa as well as olfactory-enriched forms, have been described (3Novack J.P. Charbonneau H. Bentley J.K. Walsh K.A. Beavo J.A. Biochemistry. 1991; 30: 7940-7947Crossref PubMed Scopus (39) Google Scholar, 4Charbonneau H. Kumar S. Novack J.P. Blumenthal D.K. Griffin P.R. Shabanowitz J. Hunt D.F. Beavo J.A. Walsh K.A. Biochemistry. 1991; 30: 7931-7940Crossref PubMed Scopus (67) Google Scholar, 5Bentley J.K. Kadlecek A. Sherbert C.H. Seger D. Sonnenburg W.K. Charbonneau H. Novack J.P. Beavo J.A. J. Biol. Chem. 1992; 267: 18676-18682Abstract Full Text PDF PubMed Google Scholar, 6Rossi P. Giorgi M. Geremia R. Kincaid R.L. J. Biol. Chem. 1988; 263: 15521-15527Abstract Full Text PDF PubMed Google Scholar, 7Shenolikar S. Thompson W.J. Strada S.J. Biochemistry. 1985; 24: 672-678Crossref PubMed Scopus (41) Google Scholar, 8Borisy F.F. Ronnett G.V. Cunningham A.M. Juilfs D. Beavo J. Snyder S.H. J. Neurosci. 1992; 12: 915-923Crossref PubMed Google Scholar). These CaM-PDE isozymes are expressed in distinct cell types in various tissues and have different substrate specificities, specific activities, and activation characteristics by Ca2+ and CaM (9Beavo, J., Houslay, M. D., (eds) (1990) Cyclic Nucleotide Phosphodiesterases: Structure, Function, Regulation, and Drug Action, Vol 2, p. 19, John Wiley & Sons, Chichester, United Kingdom.Google Scholar). To date, three different genes (PDE1A, PDE1B, and PDE1C) have been identified in the CaM-PDE family. PDE1A and PDE1B have been extensively characterized (5Bentley J.K. Kadlecek A. Sherbert C.H. Seger D. Sonnenburg W.K. Charbonneau H. Novack J.P. Beavo J.A. J. Biol. Chem. 1992; 267: 18676-18682Abstract Full Text PDF PubMed Google Scholar, 10Sonnenburg W.K. Seger D. Beavo J.A. J. Biol. Chem. 1993; 268: 645-652Abstract Full Text PDF PubMed Google Scholar, 11Repaske D.R. Swinnen J.V. Jin S.-L.C. Van Wyck J.J. Conti M. J. Biol. Chem. 1992; 267: 18683-18688Abstract Full Text PDF PubMed Google Scholar, 12Polli J.W. Kincaid R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11079-11083Crossref PubMed Scopus (59) Google Scholar). Two splice variants of the PDE1A gene, PDE1A1 and PDE1A2, have been isolated from bovine heart and brain, respectively (10Sonnenburg W.K. Seger D. Beavo J.A. J. Biol. Chem. 1993; 268: 645-652Abstract Full Text PDF PubMed Google Scholar, 13Sonnenburg W.K. Seger D. Kwak K.S. Huang J. Charbonneau H. Beavo J.A. J. Biol. Chem. 1995; 270: 30989-31000Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). PDE1A1 and PDE1A2 encode the bovine heart 59-kDa and bovine brain 61-kDa CaM-PDE isozymes, respectively, and differ only in their N-termini (3Novack J.P. Charbonneau H. Bentley J.K. Walsh K.A. Beavo J.A. Biochemistry. 1991; 30: 7940-7947Crossref PubMed Scopus (39) Google Scholar). PDE1B1, which encodes the bovine brain 63-kDa CaM-PDE isozyme, has only one mRNA product isolated so far (5Bentley J.K. Kadlecek A. Sherbert C.H. Seger D. Sonnenburg W.K. Charbonneau H. Novack J.P. Beavo J.A. J. Biol. Chem. 1992; 267: 18676-18682Abstract Full Text PDF PubMed Google Scholar, 11Repaske D.R. Swinnen J.V. Jin S.-L.C. Van Wyck J.J. Conti M. J. Biol. Chem. 1992; 267: 18683-18688Abstract Full Text PDF PubMed Google Scholar, 12Polli J.W. Kincaid R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11079-11083Crossref PubMed Scopus (59) Google Scholar). PDE1C is a newly identified CaM-PDE gene, and one of its products (PDE1C2) is a dominant form of CaM-PDE present in olfactory sensory neurons (14Yan C. Zhao A.Z. Bentley J.K. Loughney K. Ferguson K. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9677-9681Crossref PubMed Scopus (148) Google Scholar). The genes encoding the brain 75-kDa and testis 68-kDa CaM-PDEs have not been identified.We report here the isolation and characterization of three new splice variants of the PDE1C gene from a mouse brain library, which we call PDE1C1, PDE1C4, and PDE1C5. The protein sequences deduced from PDE1C1, PDE1C4, and PDE1C5 cDNAs are the same except in the C-terminal regions. However, they differ from the PDE1C2 protein sequence at both the N and C termini. More interestingly, PDE1C4 and PDE1C5 cDNAs have the same coding sequences but different 3′-untranslated regions (3′-UTRs), which is the first example of alternative splicing occurring solely in the 3′-UTR for mammalian PDEs. In order to understand the functional consequences of the different sequences among these variants, the kinetic properties, Ca2+ activation characteristics, inhibition by various CaM-PDE inhibitors, reciprocal inhibition between substrates, and the tissue-specific expression of multiple CaM-PDEs were systematically investigated. The data should help us understand the biological reasons for this great diversity and may provide a molecular basis for the design of selective inhibitors or activators of this PDE family.DISCUSSIONWe report here the isolation and characterization of three new mouse PDE1C cDNA clones named PDE1C1, PDE1C4, and PDE1C5. Taken together with other recent data, it now appears that alternative splicing of the PDE1C gene generates at least five distinct mRNAs: PDE1C1, PDE1C2 (14Yan C. Zhao A.Z. Bentley J.K. Loughney K. Ferguson K. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9677-9681Crossref PubMed Scopus (148) Google Scholar), PDE1C3 (26Loughney K. Martins T. Sonnenburg B. Beavo J. Ferguson K. FASEB J. 1994; 8 (abstr.): 469Crossref PubMed Scopus (0) Google Scholar), PDE1C4, and PDE1C5. Analysis of the genomic sequence of the PDE1C gene will be required to reveal the detailed mechanisms for generating these multiple species of PDE1C mRNA.PDE1C1 and PDE1C4/5 share the same N terminus, which is different from that of PDE1C2. The divergence point is located within the first calmodulin binding domain identified in PDE1A1 and PDE1A2 (3Novack J.P. Charbonneau H. Bentley J.K. Walsh K.A. Beavo J.A. Biochemistry. 1991; 30: 7940-7947Crossref PubMed Scopus (39) Google Scholar, 4Charbonneau H. Kumar S. Novack J.P. Blumenthal D.K. Griffin P.R. Shabanowitz J. Hunt D.F. Beavo J.A. Walsh K.A. Biochemistry. 1991; 30: 7931-7940Crossref PubMed Scopus (67) Google Scholar) and at a very similar relative position to the alternative splicing site that yields PDE1A1 and PDE1A2 (Fig. 1C). Similar to the differences in Ca2+ sensitivity between PDE1A1 and PDE1A2, PDE1C1 and PDE1C4/5 have much lower Ca2+ sensitivity than PDE1C2 (Fig. 3). This result further supports the idea that the N terminus of the CaM-PDE is the region where Ca2+/calmodulin binds. In addition, this PDE1C example and the PDE1A example strongly suggest that the functional differences (e.g. Ca2+ activation) among CaM-PDE isozymes can be attributed to their structural diversity and that alternative splicing provides an additional mechanism for increases in functional and structural diversity.PDE1C4 and PDE1C5 cDNAs differ only in their 3′-UTRs and thus encode the same protein. PDE1C1 and PDE1C4/5 have the same N termini and different C termini. The kinetic properties and Ca2+ sensitivity of these enzymes with different C termini are very similar, suggesting that the C-terminal sequences are not important for the enzyme's catalytic activity nor the interaction with Ca2+/calmodulin. But it is not yet known whether these different C-terminal sequences have any consequences for stability, intracellular localization, regulatory modification, or cell-specific expression. The PDE1C4 and PDE1C5 cDNAs appear to be generated by alternative splicing of 3′-UTRs. Much experimental evidence indicates that sequences in 3′-UTRs critically influence post-transcriptional regulation of mRNAs during growth, differentiation, and response to environmental stimuli (27Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3107) Google Scholar, 28Brawerman G. Cell. 1987; 48: 5-6Abstract Full Text PDF PubMed Scopus (251) Google Scholar, 29Jackson R.J. Standart N. Cell. 1990; 62: 15-24Abstract Full Text PDF PubMed Scopus (483) Google Scholar, 30Gillis P. Malter J.S. J. Biol. Chem. 1991; 266: 3172-3177Abstract Full Text PDF PubMed Google Scholar). Hence, the functional role for the alternatively spliced 3′-UTRs of PDE1C4 and PDE1C5 may be in post-transcriptional, translational, and/or tissue-specific regulation of the gene expression. Clearly, more work is required to define the functional role for the unique sequences found in the different splice variants.Although a number of compounds are capable of inhibiting the CaM-PDEs, most of them lack selectivity between CaM-PDE isozymes. The three compounds tested in this study showed some selectivity between CaM-PDE isozymes and even between splice variants. The fact that these three compounds have different selectivity profiles may reflect different mechanisms of inhibition. Availability of such selective inhibitors should not only provide useful tools for understanding biological function of each isozyme but also offer an initial starting point for development of agents having higher selectivity.CaM-PDEs with high affinities for both cAMP and cGMP have been described in various tissues, such as olfactory epithelium (8Borisy F.F. Ronnett G.V. Cunningham A.M. Juilfs D. Beavo J. Snyder S.H. J. Neurosci. 1992; 12: 915-923Crossref PubMed Google Scholar), testis (6Rossi P. Giorgi M. Geremia R. Kincaid R.L. J. Biol. Chem. 1988; 263: 15521-15527Abstract Full Text PDF PubMed Google Scholar), heart (31Reeves M.L. Leigh B.K. England P.J. Biochem. J. 1987; 241: 535-541Crossref PubMed Scopus (296) Google Scholar), and pancreas (32Vandermeers A. Vandermeers P.M.C. Rathe J. Christophe J. Biochem. J. 1983; 211: 341-347Crossref PubMed Scopus (16) Google Scholar). PDE1C2 is likely to represent the high affinity CaM-PDE activity in olfactory epithelium (8Borisy F.F. Ronnett G.V. Cunningham A.M. Juilfs D. Beavo J. Snyder S.H. J. Neurosci. 1992; 12: 915-923Crossref PubMed Google Scholar, 14Yan C. Zhao A.Z. Bentley J.K. Loughney K. Ferguson K. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9677-9681Crossref PubMed Scopus (148) Google Scholar). The data presented in this paper help to define which gene products are responsible for some of the high affinity CaM-PDE activities described in previous studies. A mouse testis enriched 68-70-kDa CaM-PDE that exhibits micromolar affinity for both cAMP and cGMP has been purified 900-fold and characterized previously (6Rossi P. Giorgi M. Geremia R. Kincaid R.L. J. Biol. Chem. 1988; 263: 15521-15527Abstract Full Text PDF PubMed Google Scholar). The present data show that PDE1C1 and PDE1C4/5 isozymes are highly expressed in the mouse testis and have high affinities to both cAMP and cGMP as well as molecular masses close to 70 kDa. These features suggest that it is very likely that some combination of these PDE1C isozymes represents the high affinity cAMP hydrolytic activity seen in the testis CaM-PDE preparation. However, two differences in biochemical parameters did not seem consistent with this conclusion. First, the purified testis enzyme is suggested to have two distinct catalytic sites, because each of the two substrates, cAMP and cGMP, acts as a noncompetitive inhibitor of the other. However, the PDE1C isozymes expressed in this study appear to have only one catalytic site for both substrates (Fig. 4). Second, the purified testis enzyme showed both high and low affinity kinetic components for both cAMP and cGMP. However, the expressed PDE1C isozymes have only the high affinity component. It seems likely that the apparent discrepancy between the expressed PDE1C activity in this study and the previously reported testis enzyme could be best explained by the assumption that the testis enzyme preparation used in the early studies may have contained more than one CaM-PDE, at least one of which has a lower affinity for cAMP and cGMP.High levels of specific PDE isozyme expression are often associated with specific functions of the region, such as the cGMP-specific PDE for visual transduction cascade in the retina (33Fung B.K. Hurley J.B. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 152-156Crossref PubMed Scopus (509) Google Scholar) and the cGMP-stimulated PDE for the aldosterone production in the adrenal cortex (34MacFarland R.T. Zelus B.D. Beavo J.A. J. Biol. Chem. 1991; 266: 136-142Abstract Full Text PDF PubMed Google Scholar). Recently, in cytosolic fractions of purified synaptosomes, the breakdown of cGMP was found to be highly stimulated by Ca2+ (35Mayer B. Klatt P. Bohme E. Schmidt K. J. Neurochem. 1992; 59: 2024-2029Crossref PubMed Scopus (98) Google Scholar). Therefore, it was proposed that a CaM-PDE is likely to be involved in preventing elevations of intracellular cGMP levels in activated, nitric oxide-producing granule cells (35Mayer B. Klatt P. Bohme E. Schmidt K. J. Neurochem. 1992; 59: 2024-2029Crossref PubMed Scopus (98) Google Scholar). It seems likely that this CaM-PDE activity may be contributed by PDE1C1 and PDE1C5. The fact that PDE1C1 and PDE1C5 represent the major CaM-PDE activities in granule cells of cerebellum suggests an important role for these PDEs in this down-regulation of cGMP of granule cells seen in response to excitatory amino acid stimulation. Primary cultured cerebellar granule cells exhibiting a high level of PDE1C activity should be a good system for further study of the physiological function of PDE1C in the granule cells.At least three CaM-PDE genes and alternative RNA processing pathways generate an unexpectedly large diversity of CaM-PDEs. More than one CaM-PDE or different splice variants are often expressed in the same tissue or even in the same cell types. Therefore, the kinetic constants or the results of inhibitor analysis determined by using biochemically purified enzymes from many tissues are often complicated by the contamination of one or more closely related enzymes. Our efforts in molecular cloning and expression of individual CaM-PDE have allowed us to define the kinetic characteristics of single isoforms. In addition, it is likely that the identification and characterization of the new PDE1C splice variants reported here and previously will aid in understanding the functional consequences resulting from unique CaM-PDE expression as well as provide molecular basis for the development of isozyme-selective CaM-PDE inhibitors. INTRODUCTIONHormones and neurotransmitters control the levels of the intracellular second messengers, cAMP and cGMP, by regulating the activity of cyclases and phosphodiesterases (PDEs). 1The abbreviations used are: PDEphosphodiesteraseCaM-PDEcalmodulin-dependent phosphodiesteraseORFopen reading frameKmMichaelis constantUTRuntranslated regionPCRpolymerase chain reactionbpbase pair(s)MOPS4-morpholinepropanesulfonic acid. At least seven different families of PDE are currently recognized (1Beavo J.A. Conti M. Heaslip R.J. Mol. Pharmacol. 1994; 46: 399-405PubMed Google Scholar, 2Beavo J.A. Physiol. Rev. 1995; 75: 725-748Crossref PubMed Scopus (1634) Google Scholar). Most families contain several distinct genes, and many of these genes encode multiple alternative splice variants in a tissue-specific manner. The facts that different PDEs have unique sequences in their catalytic and/or regulatory domains and that they are often selectively expressed in a limited number of cell types allow cell-specific regulation of cyclic nucleotide level by the PDEs. They also provides a basis for selective therapeutic intervention.The Ca2+/calmodulin-dependent PDEs (CaM-PDEs) compose one of the best known of the multiple PDE families. All CaM-PDEs are activated by calmodulin in the presence of calcium. It is thought that CaM-PDEs act as mediators between the Ca2+ and cyclic nucleotide second messenger systems that allow cyclic nucleotide-dependent processes to be regulated by increases in intracellular Ca2+ concentration (2Beavo J.A. Physiol. Rev. 1995; 75: 725-748Crossref PubMed Scopus (1634) Google Scholar). A rather large family of CaM-PDE isozymes is expressed in mammals. At least six different members, including 59-, 61-, 63-, 68-, and 75-kDa as well as olfactory-enriched forms, have been described (3Novack J.P. Charbonneau H. Bentley J.K. Walsh K.A. Beavo J.A. Biochemistry. 1991; 30: 7940-7947Crossref PubMed Scopus (39) Google Scholar, 4Charbonneau H. Kumar S. Novack J.P. Blumenthal D.K. Griffin P.R. Shabanowitz J. Hunt D.F. Beavo J.A. Walsh K.A. Biochemistry. 1991; 30: 7931-7940Crossref PubMed Scopus (67) Google Scholar, 5Bentley J.K. Kadlecek A. Sherbert C.H. Seger D. Sonnenburg W.K. Charbonneau H. Novack J.P. Beavo J.A. J. Biol. Chem. 1992; 267: 18676-18682Abstract Full Text PDF PubMed Google Scholar, 6Rossi P. Giorgi M. Geremia R. Kincaid R.L. J. Biol. Chem. 1988; 263: 15521-15527Abstract Full Text PDF PubMed Google Scholar, 7Shenolikar S. Thompson W.J. Strada S.J. Biochemistry. 1985; 24: 672-678Crossref PubMed Scopus (41) Google Scholar, 8Borisy F.F. Ronnett G.V. Cunningham A.M. Juilfs D. Beavo J. Snyder S.H. J. Neurosci. 1992; 12: 915-923Crossref PubMed Google Scholar). These CaM-PDE isozymes are expressed in distinct cell types in various tissues and have different substrate specificities, specific activities, and activation characteristics by Ca2+ and CaM (9Beavo, J., Houslay, M. D., (eds) (1990) Cyclic Nucleotide Phosphodiesterases: Structure, Function, Regulation, and Drug Action, Vol 2, p. 19, John Wiley & Sons, Chichester, United Kingdom.Google Scholar). To date, three different genes (PDE1A, PDE1B, and PDE1C) have been identified in the CaM-PDE family. PDE1A and PDE1B have been extensively characterized (5Bentley J.K. Kadlecek A. Sherbert C.H. Seger D. Sonnenburg W.K. Charbonneau H. Novack J.P. Beavo J.A. J. Biol. Chem. 1992; 267: 18676-18682Abstract Full Text PDF PubMed Google Scholar, 10Sonnenburg W.K. Seger D. Beavo J.A. J. Biol. Chem. 1993; 268: 645-652Abstract Full Text PDF PubMed Google Scholar, 11Repaske D.R. Swinnen J.V. Jin S.-L.C. Van Wyck J.J. Conti M. J. Biol. Chem. 1992; 267: 18683-18688Abstract Full Text PDF PubMed Google Scholar, 12Polli J.W. Kincaid R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11079-11083Crossref PubMed Scopus (59) Google Scholar). Two splice variants of the PDE1A gene, PDE1A1 and PDE1A2, have been isolated from bovine heart and brain, respectively (10Sonnenburg W.K. Seger D. Beavo J.A. J. Biol. Chem. 1993; 268: 645-652Abstract Full Text PDF PubMed Google Scholar, 13Sonnenburg W.K. Seger D. Kwak K.S. Huang J. Charbonneau H. Beavo J.A. J. Biol. Chem. 1995; 270: 30989-31000Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). PDE1A1 and PDE1A2 encode the bovine heart 59-kDa and bovine brain 61-kDa CaM-PDE isozymes, respectively, and differ only in their N-termini (3Novack J.P. Charbonneau H. Bentley J.K. Walsh K.A. Beavo J.A. Biochemistry. 1991; 30: 7940-7947Crossref PubMed Scopus (39) Google Scholar). PDE1B1, which encodes the bovine brain 63-kDa CaM-PDE isozyme, has only one mRNA product isolated so far (5Bentley J.K. Kadlecek A. Sherbert C.H. Seger D. Sonnenburg W.K. Charbonneau H. Novack J.P. Beavo J.A. J. Biol. Chem. 1992; 267: 18676-18682Abstract Full Text PDF PubMed Google Scholar, 11Repaske D.R. Swinnen J.V. Jin S.-L.C. Van Wyck J.J. Conti M. J. Biol. Chem. 1992; 267: 18683-18688Abstract Full Text PDF PubMed Google Scholar, 12Polli J.W. Kincaid R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11079-11083Crossref PubMed Scopus (59) Google Scholar). PDE1C is a newly identified CaM-PDE gene, and one of its products (PDE1C2) is a dominant form of CaM-PDE present in olfactory sensory neurons (14Yan C. Zhao A.Z. Bentley J.K. Loughney K. Ferguson K. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9677-9681Crossref PubMed Scopus (148) Google Scholar). The genes encoding the brain 75-kDa and testis 68-kDa CaM-PDEs have not been identified.We report here the isolation and characterization of three new splice variants of the PDE1C gene from a mouse brain library, which we call PDE1C1, PDE1C4, and PDE1C5. The protein sequences deduced from PDE1C1, PDE1C4, and PDE1C5 cDNAs are the same except in the C-terminal regions. However, they differ from the PDE1C2 protein sequence at both the N and C termini. More interestingly, PDE1C4 and PDE1C5 cDNAs have the same coding sequences but different 3′-untranslated regions (3′-UTRs), which is the first example of alternative splicing occurring solely in the 3′-UTR for mammalian PDEs. In order to understand the functional consequences of the different sequences among these variants, the kinetic properties, Ca2+ activation characteristics, inhibition by various CaM-PDE inhibitors, reciprocal inhibition between substrates, and the tissue-specific expression of multiple CaM-PDEs were systematically investigated. The data should help us understand the biological reasons for this great diversity and may provide a molecular basis for the design of selective inhibitors or activators of this PDE family.
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