Spatially distributed alternative splice variants of the renal Na-K-Cl cotransporter exhibit dramatically different affinities for the transported ions

Three splice variants of the renal Na-K-Cl cotransporter (NKCC2 F, A, and B) are spatially distributed along the thick ascending limb of the mammalian kidney. To test whether NKCC2 splice variants differ in ion transport characteristics we expressed cDNAs encoding rabbit NKCC2 F, A, and B in Xenopus oocytes and determined the ion dependence of bumetanide-sensitive (86)Rb influx. The three splice variants of NKCC2 showed dramatic differences in their kinetic behavior. The medullary variant F exhibited 3-4-fold lower affinity than variants A and B for Na(+) and K(+). Chloride affinities also markedly distinguish the three variants (K(m)F = 111.3, K(m)A = 44.7, and K(m)B = 8.9 mm Cl(-)). Thus, the kinetic properties of the NKCC2 splice variants are consistent with the spatial distribution of the variants along the thick ascending limb as they are involved in reabsorbing Na(+), K(+), and Cl(-) from a progressively diluted fluid in the tubule lumen. Variant B also showed an anomalous inhibition of rubidium influx at high extracellular Na(+) concentrations, possibly important in its highly specialized role in the macula densa. The adaptation of the kinetic characteristics of the NKCC2 variants to the luminal concentrations of substrate represents an excellent example of functional specialization and diversity that can be achieved through alternative mRNA splicing.     

Novel insights regarding the operational characteristics and teleological purpose of the renal Na+-K+-Cl2 cotransporter (NKCC2s) splice variants

The absorptive Na(+)-K(+)-Cl(-) cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H(2)O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H(2)O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2-NKCC2 interactions.     

Functional comparison of renal Na-K-Cl cotransporters between distant species

In the shark (sa), two variants of the renal Na-K-Cl cotransporter (saNKCC2A and saNKCC2F) are produced by alternative splicing of the second transmembrane domain (tm(2)). In mammals, these splice variants, as well as a third variant (NKCC2B), are spatially distributed along the thick ascending limb of Henle and exhibit divergent kinetic behaviors. To test whether different tm(2) in saNKCC2 are also associated with different kinetic phenotypes, we examined the ion dependence of (86)Rb influx for shark and rabbit splice variants expressed in Xenopus laevis oocytes. We found that, in both species, A forms have higher cation affinities than F forms. In regard to Cl affinity, however, the A-F difference was more pronounced in rabbit, and the relationship between transport activity and Cl concentration was not always sigmoidal. These results show that the tm(2) of saNKCC2 is, as in rabbit, important for Cl transport, and they suggest that the ability of the distal NKCC2-expressing segment to extract Cl from the luminal fluid differs among species. We have also found that the renal NKCC2 of distant vertebrates share similar affinities for cations. This finding points to the existence of highly conserved residues that mediate the kinetic behavior of the NKCC2 splice variants.     

Molecular mechanisms of cation transport by the renal Na+-K+-Cl- cotransporter: structural insight into the operating characteristics of the ion transport sites

Two variants of the renal Na(+)-K(+)-Cl(-) cotransporter (NKCC2), called NKCC2A and NKCC2F, display marked differences in Na(+), Rb(+), and Cl(-) affinities, yet are identical to one another except for a 23-residue membrane-associated domain that is derived from alternatively spliced exons. The proximal portion of these exons is predicted to encode the second transmembrane domain (tm2) in the form of an alpha-helix, and the distal portion, part of the following connecting segment (cs1a). In recent studies, we have taken advantage of the A-F differences in kinetic behavior to determine which regions in tm2-cs1a are involved in ion transport. Functional characterizations of chimeras in which tm2 or cs1a were interchanged between the variants showed that both regions are important in specifying ion affinities, but did not allow delineating the contribution of individual residues. Here, we have extended these structure-function analyses by studying additional mutants in which variant residues between A and F were interchanged individually in the tm2-cs1a region (amino acid number 216, 220, 223, 229, or 233 in NKCC2). None of the substitutions were found to affect K(m (C1-)), suggesting that the affinity difference for anion transport is conveyed by a combination of variant residues in this domain. However, 2 substitutions in the tm2 of F were found to affect cation constants specifically; interestingly, one of these mutations (residue 216) only affected K(m (Rb+)) while the other (residue 220) only affected K(m (Na+)). We have thus identified two novel residues in NKCC2 that play a key role in cation transport. Because such residues should be adjacent to one another on the vertical axis of the tm2 alpha-helix, our results imply, furthermore, that the ion transport sites in NKCC2 could be physically linked.     

Molecular mechanisms of Cl- transport by the renal Na(+)-K(+)-Cl- cotransporter. Identification of an intracellular locus that may form part of a high affinity Cl(-)-binding site

The 2nd transmembrane domain (tm) of the secretory Na(+)-K(+)-Cl(-) cotransporter (NKCC1) and of the kidney-specific isoform (NKCC2) has been shown to play an important role in cation transport. For NKCC2, by way of illustration, alternative splicing of exon 4, a 96-bp sequence from which tm2 is derived, leads to the formation of the NKCC2A and F variants that both exhibit unique affinities for cations. Of interest, the NKCC2 variants also exhibit substantial differences in Cl- affinity as well as in the residue composition of the first intracellular connecting segment (cs1a), which immediately follows tm2 and which too is derived from exon 4. In this study, we have prepared chimeras of the shark NKCC2A and F (saA and saF) to determine whether cs1a could play a role in Cl- transport; here, tm2 or cs1a in saF was replaced by the corresponding domain from saA (generating saA/F or saF/A, respectively). Functional analyses of these chimeras have shown that cs1a-specific residues account for most of the A-F difference in Cl- affinity. For example, Km(Cl-)s were approximately 8 mm for saF/A and saA, and approximately 70 mm for saA/F and saF. Intriguingly, variant residues in cs1a also affected cation transport; here, Km(Na+)s for the chimeras and for saA were all approximately 20 mM, and Km(Rb+) all approximately 2 mM. Regarding tm2, our studies have confirmed its importance in cation transport and have also identified novel properties for this domain. Taken together, our results demonstrate for the first time that an intracellular loop in NKCC contributes to the transport process perhaps by forming a flexible structure that positions itself between membrane spanning domains.     

A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter,NKCC1

The secretory Na-K-Cl cotransporter NKCC1 is activated by secretagogues through a phosphorylation-dependent mechanism. We found a phosphorylation stoichiometry of 3.0 +/- 0.4 phosphorylated residues/NKCC1 protein harvested from shark rectal gland tubules maximally stimulated with forskolin and calyculin A, showing that at least three sites on the cotransporter are phosphorylated upon stimulation. Three phosphoacceptor sites were identified in the N-terminal domain of the protein (at Thr(184), Thr(189), and Thr(202)) using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze tryptic fragments of the radiolabeled cotransporter. None of these residues occurs in the context of strong consensus sites for known Ser/Thr kinases. The threonines and the surrounding amino acids are highly conserved between NKCC1 and NKCC2, and similarities are also present in the Na-Cl cotransporter NCC (or TSC). This strongly suggests that the phosphoregulatory mechanism is conserved among isoforms. Through expression of shark NKCC1 mutants in HEK-293 cells, Thr(189) was found to be necessary for activation of the protein, whereas phosphorylation at Thr(184) and Thr(202) was modulatory, but not required. In conjunction with the recent finding (Darmen, R. B., Flemmer, A., and Forbush, B. (2001) J. Biol. Chem. 276, 34359-34362) that protein phosphatase-1 binds to residues 107-112 in the shark NKCC1 sequence, these results demonstrate that the N terminus of NKCC1 constitutes a phosphoregulatory domain of the transporter.     

Functional Expression of the Na-K-2Cl Cotransporter NKCC2 in Mammalian Cells Fails to Confirm the Dominant-negative Effect of the AF Splice Variant

The renal bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) is the major salt transport pathway in the apical membrane of the mammalian thick ascending limb. It is differentially spliced and the three major variants (A, B, and F) differ in their localization and transport characteristics. Most knowledge about its regulation comes from experiments in Xenopus oocytes as NKCC2 proved difficult to functionally express in a mammalian system. Here we report the cloning and functional expression of untagged and unmodified versions of the major splice variants from ferret kidney (fNKCC2A, -B, and -F) in human embryonic kidney (HEK) 293 cells. Many NKCC2 antibodies used in this study detected high molecular weight forms of the transfected proteins, probably NKCC2 dimers, but not the monomers. Interestingly, monomers were strongly detected by phosphospecific antibodies directed against phosphopeptides in the regulatory N terminus. Bumetanide-sensitive ⁸⁶Rb uptake was significantly higher in transfected HEK-293 cells and could be stimulated by incubating cells in a medium containing a low chloride concentration prior the uptake measurements. fNKCC2 was less sensitive to the reduction in chloride concentration than NKCC1. Using HEK-293 cells stably expressing fNKCC2A we also show that co-expression of variant NKCC2AF does not have the dominant-negative effect on NKCC2A activity that was seen in Xenopus oocytes, nor is it trafficked to the cell surface. In addition, fNKCC2AF is neither complex glycosylated nor phosphorylated in its N terminus regulatory region like other variants.     

A single binding motif is required for SPAK activation of the Na-K-2Cl cotransporter.

BACKGROUND: SPAK (Ste20p-related proline alanine-rich kinase) phosphorylates and activates NKCC1 (Na-K-2Cl cotransporter) in the presence of another serine/threonine kinase WNK4 (With No lysine (K)). However, whether or not the docking of SPAK to NKCC1 is a requirement for cotransporter activation has not been fully resolved. METHODS: We mutated both SPAK binding motifs in the amino-terminal tail of NKCC1 and tested the interaction between SPAK and NKCC1 using a semi in vivo yeast two-hybrid assay, (32)P-ATP in vitro phosphorylation assays, and (86)Rb(+) uptake (a K(+) congener) assays in heterologously expressed Xenopus laevis oocytes. We also used site-directed mutagenesis to identify the principle phospho-regulatory threonine residues in the amino-terminal tail of NKCC1. RESULTS: A single SPAK binding motif is necessary for isotonic NKCC1 activation. Mutation of the phenylalanine (F) residue within the motif abrogates binding and function. Phosphorylation of the cotransporter is markedly reduced in the absence of SPAK docking to NKCC1. Truncations of internal regions of the amino-terminus of NKCC1 do not disrupt protein structure enough to affect cotransporter function. Threonine residues (T(206) and T(211)) are both identified as phospho-regulatory sites of NKCC1 function. CONCLUSION: We demonstrate that physical docking of SPAK to NKCC1 is necessary for cotransporter activity under both baseline and hyperosmotic conditions. We identify T(206) and T(211) as major phospho-acceptor sites involved in cotransporter function, with T(206) common to two separate regulatory pathways: one involving SPAK, the other involving a still unknown kinase that is responsive to forskolin/PKA stimulation.     

Loop diuretic and ion-binding residues revealed by scanning mutagenesis of transmembrane helix 3 (TM3) of Na-K-Cl cotransporter (NKCC1)

The Na-K-Cl cotransporter (NKCC) plays central roles in cellular chloride homeostasis and in epithelial salt transport, but to date little is known about the mechanism by which the transporter moves ions across the membrane. We examined the functional role of transmembrane helix 3 (TM3) in NKCC1 using cysteine- and tryptophan-scanning mutagenesis and analyzed our results in the context of a structural homology model based on an alignment of NKCC1 with other amino acid polyamine organocation superfamily members, AdiC and ApcT. Mutations of residues along one face of TM3 (Tyr-383, Met-382, Ala-379, Asn-376, Ala-375, Phe-372, Gly-369, and Ile-368) had large effects on translocation rate, apparent ion affinities, and loop diuretic affinity, consistent with a proposed role of TM3 in the translocation pathway. The prediction that Met-382 is part of an extracellular gate that closes to form an occluded state is strongly supported by conformational sensitivity of this residue to 2-(trimethylammonium)ethyl methanethiosulfonate, and the bumetanide insensitivity of M382W is consistent with tryptophan blocking entry of bumetanide into the cavity. Substitution effects on residues at the intracellular end of TM3 suggest that this region is also involved in ion coordination and may be part of the translocation pathway in an inward-open conformation. Mutations of predicted pore residues had large effects on binding of bumetanide and furosemide, consistent with the hypothesis that loop diuretic drugs bind within the translocation cavity. The results presented here strongly support predictions of homology models of NKCC1 and demonstrate important roles for TM3 residues in ion translocation and loop diuretic inhibition.     

Characterization of SPAK and OSR1, regulatory kinases of the Na-K-2Cl cotransporter

Our recent studies demonstrate that SPAK (Ste20p-related Proline Alanine-rich Kinase), in combination with WNK4 [With No lysine (K) kinase], phosphorylates and stimulates the Na-K-2Cl cotransporter (NKCC1), whereas catalytically inactive SPAK (K104R) fails to activate the cotransporter. The catalytic domain of SPAK contains an activation loop between the well-conserved DFG and APE motifs. We speculated that four threonine residues (T231, T236, T243, and T247) in the activation loop might be sites of phosphorylation and kinase activation; therefore, we mutated each residue into an alanine. In this report, we demonstrate that coexpression of SPAK (T243A) or SPAK (T247A) with WNK4 not only prevented, but robustly inhibited, cotransporter activity in NKCC1-injected Xenopus laevis oocytes. These activation loop mutations produced an effect similar to that of the SPAK (K104R) mutant. In vitro phosphorylation experiments demonstrate that both intramolecular autophosphorylation of SPAK and phosphorylation of NKCC1 are significantly stronger in the presence of Mn2+ rather than Mg2+. We also show that SPAK activity is markedly inhibited by staurosporine and K252a, partially inhibited by N-ethylmaleimide and diamide, and unaffected by arsenite. OSR1, a kinase closely related to SPAK, exhibited similar kinase properties and similar functional activation of NKCC1 when coexpressed with WNK4.     

Identification of key residues involved in the dimerization of the secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1

The "secretory" Na(+)-K(+)-2Cl(-) cotransporter, NKCC1, belongs to the SLC12 gene family of electroneutral cation-chloride cotransporters. A number of these proteins, including NKCC1 itself, exist as homodimers in the membrane, suggesting that this may be a common feature of the SLC12 family. We have previously demonstrated that replacing the C-terminus of NKCC1 with that of its close homologue NKCC2 produced a fully functional chimeric protein that formed homodimers but did not dimerize with NKCC1. Here we employ a novel co-immunoprecipitation assay to study the dimerization interaction of NKCC1 using additional NKCC1/NKCC2 C-terminal chimeras and point mutants. Our results indicate that the substitution of a number of regions of the C-terminus of NKCC1 with the corresponding sequence from NKCC2 results in weakened dimerization with wild-type NKCC1, demonstrating that various residues play a role in this interaction. Most interestingly, however, we find that the replacement of a single NKCC1 residue, G812, with cysteine, the corresponding amino acid in NKCC2, results in a point mutant that displays no significant dimerization with the wild-type protein. In addition to this effect on heterodimer formation, we also find that G812 mutants can nevertheless form homodimers but that this interaction can be weaker than that observed for wild-type NKCC1. We demonstrate that our results are consistent with at least one established mechanism of protein dimer formation, that of "domain swapping", as well as with a recently reported crystal structure of the C-terminus of a bacterial SLC12 homologue.     

The Na+:Cl- cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion

The renal Na(+):Cl(-) cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl(-) influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl(-) efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr(53) and Thr(58). Elimination of these threonines together with serine residue Ser(71) completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.     

Ion transport and ligand binding by the Na-K-Cl cotransporter, structure-function studies

The cation-Cl cotransporters (CCCs) mediate the coupled movement of Na and/or K to that of Cl across the plasmalemma of animal cells. Eight CCCs have been identified to date: two Na-K-Cl cotransporters (NKCC), four K-Cl cotransporters (KCCs), one Na-Cl cotransporter (NCC) and one CCC interacting protein (CIP). All of the NKCCs and KCCs are inhibited by loop diuretics; mercury and other modifying agents are also known to block NKCC-mediated transport. In this work, we have utilized a mutational approach to study the interaction between different substrates and the NKCCs. We relied on the strategy of exchanging domains between functionally distinct carriers (the shark NKCCl and the human NKCCl) to identify residues or group of residues that are involved in the interaction with ions, loop diuretics and Hg. Our results show that the N- and C-termini have no role in determining the species differences in ion transport and bumetanide binding. On the other hand, the interaction between Hg and the NKCCs is found to partially involve the C-terminus through residues that contain available sulfhydryl groups. Within the transmembrane segments, variant residues in the 2nd, 4th and 7th predicted alpha-helices are shown to encode the differences in ion transport between the shark and the human cotransporters. For loop diuretic binding, several regions throughout the central domain appear to be involved. Interestingly, these regions are not the same as those involved in cation or anion transport, and in Hg binding.     

Genomic organization,chromosomal localization,and alternative splicing of the human phosphodiesterase 8B gene

We have characterized the gene for human phosphodiesterase 8B, PDE8B, and cloned the full-length cDNA for human PDE8B (PDE8B1) and two splice variants (PDE8B2 and PDE8B3). The PDE8B gene is mapped to the long arm of chromosome 5 (5q13) and is composed of 22 exons spanning over approximately 200kb. The donor and acceptor splice site sequences match the consensus sequences for the exon-intron boundaries of most eukaryotic genes. PDE8B1 encodes an 885 amino acid enzyme, containing an N-terminal REC domain, a PAS domain, and a C-terminal catalytic domain. PDE8B2 and PDE8B3 both have deletion in the PAS domain and encode 838 and 788 amino acid proteins, respectively. RT-PCR analysis revealed that while PDE8B1 is the most abundant variant in thyroid gland, PDE8B3, but not PDE8B1, is the most abundant form in brain. These findings suggest that selective usage of exons produces three different PDE8B variants that exhibit a tissue-specific expression pattern.     

Na(+) -K(+) -2Cl(-) cotransporter type 2 trafficking and activity: The role of interacting proteins

The central role of Na(+) -K(+) -2Cl(-) cotransporter type 2 (NKCC2) in vectorial transepithelial salt reabsorption in thick ascending limb cells from Henle's loop in the kidney is evidenced by the effects of loop diuretics, the pharmacological inhibitors of NKCC2, that are amongst the most powerful antihypertensive drugs available to date. Moreover, genetic mutations of the NKCC2 encoding gene resulting in impaired apical targeting and function of NKCC2 transporter give rise to a pathological phenotype known as type I Bartter syndrome, characterised by a severe volume depletion, hypokalaemia and metabolic alkalosis with high prenatal mortality. On the contrary, excessive NKCC2 activity has been linked with inherited hypertension in humans and in rodent models. Interestingly, in animal models of hypertension, NKCC2 upregulation is achieved by post-translational mechanisms underlining the need to analyse the molecular mechanisms involved in the regulation of NKCC2 trafficking and activity to gain insights in the pathogenesis of hypertension.     

Structure-function relationships of the human bitter taste receptor hTAS2R1: insights from molecular modeling studies

Bitter taste receptors (T2Rs) belong to G-protein-coupled receptors (GPCRs). Despite extensive studies, the precise mechanisms of GPCR activation are still poorly understood. In this study, the models of the human bitter taste receptor hTAS2R1 alone and in complex with various ligands were constructed on the basis of template-based modeling and molecular docking. Then these models were subjected to all-atom molecular dynamics (MD) simulations in explicit lipid bilayers. The binding pocket of hTAS2R1 is mainly formed by transmembrane helix (TM) III, TM V, TM VI, and TM VII. Most of the residues contributing to ligand binding are positionally conserved comparing with other hTAS2Rs. By comparing the final conformations obtained by extensive MD simulations, we identified the changes in the transmembrane helices and the intra- and extracellular loops, which were expected to initiate the activation of the receptor. The intracellular loop II (ICL2) and TM III were found to play prominent roles in the process of activation. We proposed that a set of interactions between the aromatic Phe115 in the middle of ICL2 and three residues (Tyr103, Lys106, and Val107) at the cytoplasmic end of TM III may serve as a conformational switch of hTAS2R1 activation. All of the residues involved in the switch are highly conserved among T2Rs. This indicates that the control switch we proposed may be universal in T2Rs. Besides, our results also suggest that the formation of a short helical segment in ICL2 may be necessary for the activation of hTAS2R1.     

Activation of the Na-K-Cl cotransporter NKCC1 detected with a phospho-specific antibody

The Na-K-Cl cotransporter NKCC1 is activated by phosphorylation of a regulatory domain in its N terminus. In the accompanying paper (Darman, R. B., and Forbush, B. (2002) J. Biol. Chem. 277, 37542-37550), we identify three phosphothreonines important in this process. Using a phospho-specific antibody (anti-phospho-NKCC1 antibody R5) raised against a diphosphopeptide containing Thr(212) and Thr(217) of human NKCC, we were readily able to monitor the cotransporter activation state. In (32)P phosphorylation experiments with rectal gland tubules, we show that the R5 antibody signal is proportional to the amount of (32)P incorporated into NKCC1; and in experiments with NKCC1-transfected HEK-293 cells, we demonstrate that R5-detected phosphorylation directly mirrors functional activation. Immunofluorescence analysis of shark rectal gland shows activation-dependent R5 antibody staining along the basolateral membrane. In perfused rat parotid glands, isoproterenol induced staining of both acinar and ductal cells along the basolateral membrane. Isoproterenol also induced basolateral staining of the epithelial cells in rat trachea, whereas basal cells in the subepithelial tissue displayed heavy, non-polarized staining of the cell membrane. In rat colon, agonist stimulation induced staining along the basolateral membrane of crypt cells. These data provide direct evidence of NKCC1 regulation in these tissues, and they further link phosphorylation of NKCC1 with its activation in transfected cells and native tissue. The high conservation of the regulatory threonine residues among NKCC1, NKCC2, and NCC family members, together with the fact that tissues from divergent vertebrate species exhibit similar R5-binding profiles, lends further support to the role of this regulatory locus in vivo.     

Isolation and differential tissue distribution of two human cDNAs encoding PDE1 splice variants.

A cDNA selection technique has been used to isolate full-length human cDNAs of the phosphodiesterase 1 (PDE1) calcium calmodulin (CaM)-regulated phosphodiesterase gene family. We isolated cDNAs representing multiple splice variants of PDE1A, 1B and 1C from a variety of tissues. Included among these were two novel splice variants for PDE1A and 1B. The first, PDE1A5, encodes a 519-residue protein, which is different from PDE1A1 by the insertion of 14 residues, a divergent carboxy terminus and also differs from PDE1A3 through a divergent amino terminus. Our second novel splice variant represents the first occurrence of a splice variant of the PDE1B gene. PDE1B2 encodes a 516-residue protein and diverges from PDE1B1 by the replacement of the first 38 residues by an alternative 18, which is predicted to be functionally significant. Using the splice variant sequence differences to perform comparative Northern analysis, we have demonstrated that each variant has a differential tissue distribution.