Stage and cell-specific expression of calmodulin-dependent phosphodiesterases in mouse testis.

Calcium and cyclic nucleotides are second messengers that regulate the development and functional activity of spermatozoa. Calcium/calmodulin-dependent phosphodiesterases (CaM-PDEs) are abundant in testicular cells and in mature spermatozoa and provide one means by which calcium regulates cellular cyclic nucleotide content. We examined the spatial and temporal expression profiles of three knownCaM-PDE genes, PDE1A, PDE1B, and PDE1C, in the testis. In situ hybridization and immunofluorescent staining showed that both PDE1A and PDE1C are highly expressed but at different stages in developing germ cells. However, a very low hybridization signal of PDE1B exists uniformly throughout the seminiferous epithelium and the interstitium. More specifically, PDE1A mRNA is found in round to elongated spermatids, with protein expression in the tails of elongated and maturing spermatids. In contrast, PDE1C mRNA accumulates during early meiotic prophase and throughout meiotic and postmeiotic stages. Immunocytochemistry showed a diffuse, presumably cytosolic distribution of the expressed protein. The distinct spatial and temporal expression patterns of CaM-PDEs suggest important but different physiological roles for these CaM-PDEs in developing and mature spermatozoa.

Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase.

Human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase (PDE5A) cDNAs were isolated. A 3.1-kb composite DNA sequence assembled from overlapping cDNAs encodes an 875-amino-acid protein with a predicted molecular mass of 100012 Da (PDE5A1). Extracts prepared from yeast expressing human PDE5A1 hydrolyzed cGMP. This activity was inhibited by the selective PDE5 inhibitors zaprinast and DMPPO. PDE5A mRNA is expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung. A 5'-splice variant, PDE5A2, encodes an 833-amino-acid protein with eight unique amino acids at the amino terminus. PDE5A maps to chromosome 4q 25-27.

Identification, quantitation, and cellular localization of PDE1 calmodulin-stimulated cyclic nucleotide phosphodiesterases.

The calmodulin-stimulated cyclic nucleotide phosphodiesterases (PDE1s) constitute a large gene family and are found in a wide variety of tissues and cells. Because of the functional diversity of PDE1 genes and the observation that these isozymes often make up a major component of the total cyclic nucleotide hydrolytic activity in certain cell types, PDE1s are of growing interest as targets for therapeutic intervention. Here we describe a series of methodologies to identify, quantitate, and determine the cellular expression of PDE1 isozymes. We describe first the resolution of different PDEs using high-performance anion-exchange chromatography and then a Western blotting methodology for identifying or authenticating PDE1 activities. Next we present an immunoprecipitation method that can be used for quantitating specific PDE1 isoforms and describe the use of RNase protection analysis for further identification of PDE1 subtypes. Finally, we provide a simple, immunocytochemical method for determining the cellular expression of PDE1 isozymes. Combined, the above methodologies should allow an investigator to identify, quantitate, and determine the cellular localization of PDE1 isozymes in any tissue with little ambiguity.

Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype.

The diversity among cyclic nucleotide phosphodiesterases provides multiple mechanisms for regulation of cAMP and cGMP in the cardiovascular system. Here we report that a calmodulin-stimulated phosphodiesterase (PDE1C) is highly expressed in proliferating human arterial smooth muscle cells (SMCs) in primary culture, but not in the quiescent SMCs of intact human aorta. High levels of PDE1C were found in primary cultures of SMCs derived from explants of human newborn and adult aortas, and in SMCs cultured from severe atherosclerotic lesions. PDE1C was the major cAMP hydrolytic activity in these SMCs. PDE expression patterns in primary SMC cultures from monkey and rat aortas were different from those from human cells. In monkey, high expression of PDE1B was found, whereas PDE1C was not detected. In rat SMCs, PDE1A was the only detectable calmodulin-stimulated PDE. These findings suggest that many of the commonly used animal species may not provide good models for studying the roles of PDEs in proliferation of human SMCs. More importantly, the observation that PDE1C is induced only in proliferating SMCs suggests that it may be both an indicator of proliferation and a possible target for treatment of atherosclerosis or restenosis after angioplasty, conditions in which proliferation of arterial SMCs is negatively modulated by cyclic nucleotides.

Isolation and characterization of human cDNAs encoding a cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase.

Human cyclic GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2A3) cDNAs were cloned from hippocampus and fetal brain cDNA libraries. A 4.2-kb composite DNA sequence constructed from overlapping cDNA clones encodes a 941 amino acid protein with a predicted molecular mass of 105,715 Da. Extracts prepared from yeast expressing the human PDE2A3 hydrolyzed both cyclic AMP (cAMP) and cyclic GMP (cGMP). This activity was inhibited by EHNA, a selective PDE2 inhibitor, and was stimulated three-fold by cGMP. Human PDE2A is expressed in brain and to a lesser extent in heart, placenta, lung, skeletal muscle, kidney and pancreas. The human PDE2A3 differs from the bovine PDE2A1 and rat PDE2A2 proteins at the amino terminus but its amino-terminal sequence is identical to the bovine PDE2A3 sequence. The different amino termini probably arise from alternative exon splicing of the PDE2A mRNA.

Isolation and characterization of cDNAs corresponding to two human calcium, calmodulin-regulated, 3',5'-cyclic nucleotide phosphodiesterases.

cDNAs corresponding to two human calcium, calmodulin (CaM)-regulated 3',5'-cyclic nucleotide phosphodiesterases (PDEs) were isolated. One, Hcam1 (PDE1A3), corresponds to the bovine 61-kDa CaM PDE (PDE1A2). The second, Hcam3 (PDE1C), represents a novel phosphodiesterase gene. Hcam1 encodes a 535-amino acid protein that differs most notably from the bovine 61-kDa CaM PDE by the presence of a 14-amino acid insertion and a divergent carboxyl terminus. RNase protection studies indicated that Hcam1 is represented in human RNA from several tissues, including brain, kidney, testes, and heart. Two carboxyl-terminal splice variants for Hcam3 were isolated. One, Hcam3b (PDE1C1), encodes a protein 634 amino acids (72 kDa) in length. The other, Hcam3a (PDE1C3), diverges from Hcam3b 4 amino acids from the carboxyl terminus of Hcam3b, and extends an additional 79 amino acids. All the cDNAs isolated for Hcam3a are incomplete; they do not include the 5'-end of the open reading frame. Northern analysis revealed that both splice variants were expressed in several tissues, including brain and heart, and that there may be additional splice variants. Amino-truncated recombinant proteins were expressed in yeast and characterized biochemically. Hcam3a has a high affinity for both cAMP and cGMP and thus has distinctly different kinetic parameters from Hcam1, which has a higher affinity for cGMP than for cAMP. Both PDE1C enzymes were inhibited by isobutylmethylxanthine, 8-methoxymethyl isobutylmethylxanthine, zaprinast, and vinpocetine.

Identification of inhibitory and calmodulin-binding domains of the PDE1A1 and PDE1A2 calmodulin-stimulated cyclic nucleotide phosphodiesterases.

Using a bovine 61-kDa (PDE1A2) calmodulin-stimulated phosphodiesterase (CaM-PDE) cDNA and a bovine lung 59-kDa (PDE1A1) CaM-PDE cDNA reported here, we have identified two new regions within the primary structure of these two related isozymes that are important for regulation by Ca2+/CaM. PDE1A1 is identical to the PDE1A2 isozyme except for the amino-terminal 18 residues. In agreement with earlier studies, the CaM concentration required for half-maximal activation (KCaM) of recombinant PDE1A1 (0.3 nM) was approximately 10-fold less than the KCaM for recombinant PDE1A2 (4 nM). A series of deletion mutations of the PDE1A2 cDNA removing nucleotide sequence encoding the first 46-106 aminoterminal residues were constructed and expressed using the baculovirus system. Deletion of the amino acids encompassing a previously identified, putative CaM-binding domain (residues 4-46) produced a polypeptide that was still activated 3-fold by CaM (KCaM approximately 3 nM). However, complete CaM-independent activation occurred when residues 4-98 were deleted. To determine the location of the additional CaM-binding domain(s), the inhibitory potency of seven overlapping, synthetic peptides spanning amino acids 76-149 of PDE1A2 was tested using the CaM-activated enzyme. One peptide spanning amino acids 114-137 of PDE1A2 appeared to be the most potent inhibitor of CaM-stimulated activity. These results reveal the existence of a CaM-binding domain located approximately 90 residues carboxyl-terminal to the putative CaM-binding domains previously identified within the PDE1A1 and PDE1A2 isozymes. Moreover, a discrete segment important for holding these CaM-PDEs in a less active state at low Ca2+ concentrations is located between the two CaM-binding domains.

An essential aspartic acid at each of two allosteric cGMP-binding sites of a cGMP-specific phosphodiesterase.

The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80-90 residues in length and are arranged in tandem within the putative cGMP-binding domains. In the bovine lung cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span residues 228-311 (a) and 410-500 (b). An aspartic acid (residue 289 or 478) that is invariant in repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with approximately 1.8 microM cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (Kd > 10 microM) and the D478A mutant showed increased affinity for cGMP (Kd approximately 0.5 microM) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (koff = 0.26 h-1) and rapidly dissociating (koff = 1.00 h-1) sites of cGMP binding. In contrast, the D289A mutant displayed a single koff = 1.24 h-1, which was similar to the calculated koff for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single koff = 0.29 h-1, which was similar to that calculated for the slow site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site.

Phosphorylation of the 61-kDa calmodulin-stimulated cyclic nucleotide phosphodiesterase at serine 120 reduces its affinity for calmodulin.

Phosphorylation of the 61-kDa isoform of bovine calmodulin (CaM)-stimulated cyclic nucleotide phosphodiesterase (CaM-PDE) by the catalytic subunit of cyclic AMP-dependent protein kinase A (PKA) results in a decrease in the affinity of the enzyme for calmodulin [Sharma, R. K., & Wang, J. H. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 2603-2607]. In the present study, purified 61-kDa CaM-PDE was phosphorylated in the presence of [gamma-32P]ATP and cleaved with a Lys-C endoproteinase. The resultant phosphopeptides were resolved by reverse-phase HPLC and analyzed by electrospray mass spectrometry and Edman sequencing. Serine residues 120 and 138 were identified as the principal sites of phosphorylation. A cDNA encoding the 61-kDa CaM-PDE [Sonnenburg, W. K., Seger, D., & Beavo, J. A. (1993) J. Biol. Chem. 268, 645-652] was used to generate point mutants in which either or both of these serines were replaced with alanine. The mutants were expressed in COS-7 cells, purified, and phosphorylated. Phosphorylation of the mutant Ser 138-->Ala resulted in a decrease in affinity for CaM that was comparable to that seen with the wild-type enzyme. In contrast, phosphorylation of the mutant Ser 120-->Ala had virtually no effect on CaM affinity. We conclude that phosphorylation of serine 120 by PKA is responsible for the reduction in affinity of the 61-kDa CaM-PDE for CaM.

Differential expression of the 61 kDa and 63 kDa calmodulin-dependent phosphodiesterases in the mouse brain.

Based on their relative abundance and regulation by Ca2+ and by phosphorylation in vitro, it is thought that the Ca2+/calmodulin-dependent phosphodiesterases (CaM-PDEs) are important modulators of cyclic nucleotide function in the brain. Two of the most abundant CaM-PDEs in the brain are the 61 kDa and 63 kDa isozymes. In this study, the regional and cellular expression of mRNA encoding these two different isoforms in mouse brain has been determined by in situ hybridization. The 63 kDa CaM-PDE mRNA has a wide-spread but uneven distribution. Very strong hybridization signals are present in the caudate-putamen, nucleus accumbens, olfactory tubercle, and dentate gyrus of the hippocampus. Somewhat lesser amounts of 63 kDa CaM-PDE mRNA are present in the olfactory bulb and piriform cortex. Weaker but still easily discernible hybridization signals are seen in several layers of the cerebral cortex, CA1 and CA3 regions of the hippocampus, amygdaloid nuclear complex, thalamus, hypothalamus, midbrain, brainstem, cerebellum, and spinal cord. A weak hybridization signal was detected in the globus pallidus of the basal ganglia. In general, the distribution of the 63 kDa CaM-PDE is very similar to that of dopamine receptors, suggesting that it may modulate dopamine function. In contrast, the 61 kDa CaM-PDE mRNA has a more limited and much different distribution, with the highest level of expression in the cerebral cortex and in the pyramidal cells of the hippocampus. A moderate hybridization signal was detected in the medial habenula and amygdaloid nuclear complex. In addition, small subsets of neurons in several other regions showed specific hybridization. Both PDE mRNAs appear to be localized exclusively in neuronal cell bodies. Their distinct distribution suggests important but different physiological roles for these two isozymes in the regional regulation of cyclic nucleotides in the CNS. Since these two isozymes are differentially phosphorylated by cAMP-dependent and Ca2+/CaM-dependent protein kinases, the differential expression also provides a potential mechanism by which these PDEs can differentially regulate cAMP and cGMP in different brain areas. The high expression levels in specific subsets of neurons also suggest that agents increasing Ca2+ in these neurons will increase the rate of cyclic nucleotide degradation.

Cyclic GMP and regulation of cyclic nucleotide hydrolysis.

Several of the different PDE isozyme families have the ability in vitro to hydrolyze cGMP. In particular they include the CaM-dependent PDEs, the cGMP-stimulated PDEs, and the cGMP binding, cGMP-specific PDEs. Existing evidence suggests or demonstrates that in different cell types, each of these can be important determinants for the control of cGMP steady-state levels. Each of these enzymes is differentially expressed and regulated; moreover, the amount of the enzyme expressed and the mode of regulation determine to a large extent the rate of rise, maximal level, rate of fall, and duration of the cGMP signal in the cell. In addition to enzymes that function to degrade cGMP at least two also are regulated by cGMP both in vitro and in the intact cell. The cGMP-stimulated PDE has the ability to decrease cAMP levels in response to cGMP and the cGMP-inhibited PDE can increase cAMP levels in response to cGMP. We are just beginning to define how many different isozymes of PDE exist in mammalian tissues, where they are located, and how they are regulated. Selective inhibitors to each are being developed and studies designed to define structural features that determine the mechanisms of action and regulation of the PDEs have been initiated. It is expected that in the next few years more PDEs will be discovered and the functions of the new an existing ones with be more clearly defined.

The role of protein phosphorylation in the regulation of cyclic nucleotide phosphodiesterases.

The cyclic nucleotide phosphodiesterases constitute a complex superfamily of enzymes responsible for catalyzing the hydrolysis of cyclic nucleotides. Regulation of cyclic nucleotide phosphodiesterases is one of the two major mechanisms by which intracellular cyclic nucleotide levels are controlled. In many cases the fluctuations in cyclic nucleotide levels in response to hormones is due to the hormone responsiveness of the phosphodiesterase. Isozymes of the cGMP-inhibited, cAMP-specific, calmodulin-stimulated and cGMP-binding phosphodiesterases have been demonstrated to be substrates for protein kinases. Here we review the evidence that hormonally responsive phosphorylation acts to regulate cyclic nucleotide phosphodiesterases. In particular, the cGMP-inhibited phosphodiesterases, which can be phosphorylated by at least two different protein kinases, are activated as a result of phosphorylation. In contrast, phosphorylation of the calmodulin-stimulated phosphodiesterases, which coincides with a decreased sensitivity to activation by calmodulin, results in decreased phosphodiesterase activity.

The structure of a bovine lung cGMP-binding, cGMP-specific phosphodiesterase deduced from a cDNA clone.

Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucleotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair PCR product was recovered and used as a probe to screen a bovine lung cDNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB-PDE was isolated. The open reading frame of this cDNA predicted an 875 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cGB-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE contained a segment (AA 578-812) that was homologous to the putative catalytic region conserved among all mammalian PDEs and a segment (AA 142-526) that was homologous to the putative cGMP binding region of the cGMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the putative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein kinase.

Molecular cloning of a cDNA encoding the "61-kDa" calmodulin-stimulated cyclic nucleotide phosphodiesterase. Tissue-specific expression of structurally related isoforms.

We have isolated a 2287-bp cDNA encoding the 61-kDa calmodulin-stimulated cyclic nucleotide phosphodiesterase (CaM PDE) from a bovine brain library. A large open reading frame within the cDNA encodes a 530-residue polypeptide which is identical to the sequence of the purified protein previously determined by direct amino acid sequencing. Moreover, COS cells transfected with the cDNA express a cAMP and cGMP hydrolytic activity that is stimulated by calcium and calmodulin, confirming that the cDNA represents a mRNA species encoding a CaM PDE isozyme. RNase protection analyses indicate that either 61-kDa CaM PDE mRNA or structurally related transcripts encoding different CaM PDE isoforms are expressed in a tissue-specific manner. Total RNA isolated from brain (cerebral cortex, basal ganglia, hippocampus, cerebellum, and medulla/spinal cord), heart, aorta, liver, kidney outer medulla, kidney papilla, trachea, and lung completely protected a 410-base antisense riboprobe corresponding to sequence encoding a portion of the catalytic domain. Little or no protection was detected using adrenal cortex, adrenal medulla, liver, kidney cortex, spleen, or T-lymphocyte total RNA. Only brain RNA completely protected a 240-base antisense riboprobe corresponding to the 61-kDa CaM PDE amino terminus encompassing a putative calmodulin-binding domain. However, heart, aorta, liver, kidney, trachea, and lung RNA protected 150 bases of this riboprobe suggesting that these tissues express an isoform structurally related to the 61-kDa CaM PDE. Northern analysis of mRNA isolated from brain, heart, aorta, liver, kidney, lung, and trachea revealed that the cDNA hybridizes with a 3.8- and a 4.4-kb (kilobase) mRNA species. Interestingly, Northern blots of bovine cerebral cortex and heart mRNA probed under stringent conditions with antisense transcripts corresponding to either the 5'- or 3'-untranslated sequence of the 61-kDa CaM PDE cDNA hybridized with only the 4.4-kb mRNA from both tissues. Since different, yet structurally similar CaM PDE isoforms are expressed in brain and in heart, this result, in addition to the RNase protection data, is consistent with the idea that the mRNAs encoding these two CaM PDE isoforms are products of an alternately spliced gene.

Molecular cloning of cDNA encoding a "63"-kDa calmodulin-stimulated phosphodiesterase from bovine brain.

Partially degenerate oligonucleotides based on peptide sequence were used to isolate cDNA to a 63-kDa bovine brain calmodulin-stimulated phosphodiesterase (CaM-PDE) isozyme. A 412-base pair polymerase chain reaction fragment was obtained and used along with the oligonucleotides to isolate several cDNAs each encoding sequence identical to known peptide sequences from the 63-kDa CaM-PDE. The largest cDNA contained a full-length open reading frame (ORF) encoding a 534 amino acid, 61,005-dalton protein. It had 59% amino acid identity to the 61-kDa bovine brain CaM-PDE and included a carboxyl-terminal conserved domain containing the PDE catalytic domain consensus sequences. The NH2-terminal region fits the criteria for a calmodulin-binding domain. When its expression was driven by a cytomegalovirus promoter on a pCDM8 vector in COS-7 cells, the cDNA encoded a catalytically active, calmodulin-stimulated PDE. Northern analysis of RNA from several tissues with a probe containing much of the conserved PDE catalytic domain showed only a single band of 4.0 kilobases. Hybridization was seen in mRNA from several regions of the central nervous system with the greatest signal in basal ganglia. Strong signals also were seen in other tissues including kidney papilla and adrenal medulla. Antisense RNA probes were used in RNase-protection assays to look for evidence of multiple 63-kDa CaM-PDE transcripts. A catalytic domain probe was fully protected by RNA from cerebral cortex, basal ganglia, cerebellum, hippocampus, adrenal medulla, and kidney papilla. However, a probe to the NH2-terminal region was fully protected only by brain and adrenal medullary RNA indicating the likelihood of one or more isozyme(s) divergent in this region in the kidney papilla.

Molecular cloning of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase cDNA. Identification and distribution of isozyme variants.

We have cloned a 4.2-kilobase pair (kb) cDNA that encodes the cyclic GMP-stimulated phosphodiesterase (cGS PDE) from a bovine adrenal cortex library. The 921-residue polypeptide deduced from the cDNA nucleotide sequence is nearly identical with the complete amino acid sequence of the cGS PDE purified from a soluble bovine heart extract. Moreover, PPD-S49 cells transfected with the cGS PDE cDNA express a soluble cAMP hydrolytic activity that is enhanced by cGMP. Total RNA isolated from several bovine tissues were screened for cGS PDE transcript by Northern blot analysis. The cGS PDE cDNA appears to hybridize to a single 4.5-4.6-kb mRNA species. Although the cGS PDE mRNA is most abundant in the adrenal cortex, it is also concentrated in the adrenal medulla and heart and in anatomically distinct regions of the brain and kidney. A mRNA species encoding a putative variant cGS PDE isoform was detected by RNase protection. Total RNA isolated from adrenal cortex, adrenal medulla, liver, kidney, trachea, lung, spleen, and T-lymphocytes completely protected a 452-base riboprobe encoding 100 residues of the adrenal cortex cGS PDE amino terminus. In contrast, RNAs isolated from brain (cerebral cortex, hippocampus, and basal ganglia) protected only 268 bases of this riboprobe. The RNase protection pattern of this same probe using heart RNA showed major bands at both 268 and 452 bases, suggesting that two different cGS PDE mRNA species are expressed. These results indicate that the cGS PDE is widely expressed in a variety of tissues. Moreover, these studies suggest that at least one different cGS PDE isoform having a structurally distinct amino-terminal domain is expressed in brain and heart.

Amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase from bovine heart.

The complete amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase (cGS-PDE) of bovine heart has been determined by analysis of five digests of the protein; placement of the C-terminal 330 residues has been confirmed by interpretation of the corresponding partial cDNA clone. The holoenzyme is a homodimer of two identical N alpha-acetylated polypeptide chains of 921 residues, each with a calculated molecular weight of 103,244. The C-terminal region, residues 613-871, of the cGS-PDE comprises a catalytic domain that is conserved in all phosphodiesterase sequences except those of PDE 1 from Saccharomyces cerevisiae and a secreted PDE from Dictyostelium. A second conserved region, residues 209-567, is homologous to corresponding regions of the alpha and alpha' subunits of the photoreceptor phosphodiesterases. This conserved domain specifically binds cGMP and is involved in the allosteric regulation of the cGS-PDE. This regulatory domain contains two tandem, internal repeats, suggesting that it evolved from an ancestral gene duplication. Common cyclic nucleotide binding properties and a distant structural relationship provide evidence that the catalytic and regulatory domains within the cGS- and photoreceptor PDEs are also related by an ancient internal gene duplication.

A prostaglandin E receptor coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rabbit cortical collecting tubule cells.

At different concentrations, prostaglandin E2 (PGE2) can either stimulate or inhibit cAMP formation in freshly isolated rabbit cortical collecting tubule (RCCT) cells, but in cultured RCCT cells PGE2 can only stimulate cAMP synthesis (Sonnenburg, W. K., and Smith W. L. (1989) J. Biol. Chem. 263, 6155-6160). Here, we report characteristics of [3H]PGE2 binding to membrane receptor preparations from both freshly isolated and cultured RCCT cells. [3H]PGE2 binding to membranes from freshly isolated RCCT cells was saturable and partially reversible. Equilibrium binding analyses indicated that in the absence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) there is a single class of PGE2 binding sites (KD = 4.2 +/- 0.4 nM; Bmax = 583 +/- 28 fmol/mg); in the presence of 100 microM GTP gamma S, there is also only one class of binding sites but with a somewhat lower KD = 1.2 +/- 0.5 nM (Bmax = 370 +/- 40 fmol/mg). This stimulatory effect of GTP gamma S was blocked by pretreatment of the freshly isolated RCCT cells with pertussis toxin. The relative affinities of prostanoids for the [3H]PGE2-binding site were determined to be 17,18,19,20-tetranor-16-phenoxy-PGE2-methylsulfonylamide (sulprostone) approximately PGE2 approximately PGE1 approximately 16,16-dimethyl-PGE2 greater than carbacyclin approximately PGF2 alpha greater than PGD2. This is the order of potency with which prostaglandins inhibit arginine vasopressin-induced cAMP formation in fresh RCCT cells. Interestingly, [3H]PGE2 binding to membranes from cultured cells, which, unlike fresh cells, fail to show an inhibitory response to PGE2, was only 10-20% of that observed with membranes from fresh cells; moreover, binding of [3H]PGE2 to membranes from cultured cells was neither stimulated by GTP gamma S nor inhibited by sulprostone. The prostanoid binding specificities and the unusual pertussis toxin-sensitive, stimulatory effect of GTP gamma S on binding of [3H]PGE2 to membranes from freshly isolated RCCT cells are characteristics shared by a Gi-linked PGE receptor from renal medulla (Watanabe, T., Umegaki, K., and Smith, W. L. (1986) J. Biol. Chem. 261, 14340-14349). Our results suggest that the [3H]PGE2 binding site of freshly isolated RCCT cells is the PGE receptor which is coupled to a Gi to attenuate arginine vasopressin-induced cAMP synthesis in the renal collecting tubule.

Identification of a noncatalytic cGMP-binding domain conserved in both the cGMP-stimulated and photoreceptor cyclic nucleotide phosphodiesterases.

Partial amino acid sequence has been determined for the cone, alpha' subunit of the bovine photoreceptor cyclic nucleotide phosphodiesterase (PDE) and deduced from nucleotide sequences of a partial cDNA clone. These sequences identify the alpha' subunit as the product of a gene that is distinct from those encoding the alpha or beta subunits of the membrane-associated rod photoreceptor PDE. Comparisons between the recently determined cGMP-stimulated-PDE sequence and those of the alpha and alpha' photoreceptor PDE subunits reveal an unexpected sequence similarity. In addition to the catalytic domain conserved in eukaryotic PDEs, all three PDEs possess a second conserved segment of approximately 340 residues that contains two internally homologous repeats. Limited proteolysis and direct photolabeling studies indicate that the noncatalytic, cGMP-binding site(s) in the cGMP-stimulated PDE is located within this conserved domain, suggesting that it also may serve this function in the photoreceptor PDEs. Moreover, other PDEs that do not bind cGMP at noncatalytic sites do not contain this conserved domain. The function of the conserved segment in the photoreceptor PDEs is not known, but the homology to allosteric sites of the cGMP-stimulated PDE suggests a role in cGMP binding and modulation of enzyme activity.