(cAMP/Ca2+/smell/tissue-specific expression). CHEN YAN*, ALLEN Z. ZHAO*, J. KELLEY BENTLEY*, KATE LOUGHNEYt, KEN FERGUSONt, AND JOSEPH A.
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9677-9681, October 1995 Neurobiology
Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons (cAMP/Ca2+/smell/tissue-specific expression)
CHEN YAN*, ALLEN Z. ZHAO*, J. KELLEY BENTLEY*, KATE LOUGHNEYt, KEN FERGUSONt, AND JOSEPH A. BEAVO*t *Department of Pharmacology, University of Washington, Seattle, WA 98195; and tlcos Corporation, 22021-20th Avenue S.E., Bothell, WA 98021 Communicated by William A. Catterall, University of Washington School of Medicine, Seattle, WA, June 26, 1995
suggesting that th, Ca2+/calmodulin-dependent PDE (CaMPDE) is quite active under these conditions (15, 16). Finally, Ca2+ appears to be a regulator in the control of cAMP signal termination. For example, electrophysiological studies show a negative correlation between cAMP and cytosolic Ca2+ levels. Recordings from olfactory neurons in Ca2+-free medium show an increased amplitude and duration of response to odorants (17, 18). An analogous regulation by Ca2+ occurs in photoreceptors, where Ca2+ regulates termination of the light response by stimulation of guanylyl cyclase activity (19). This is consistent with the idea that the elevated Ca2+ level after odorant stimulation (20, 21) provides an important control for signal termination via stimulation of CaM-PDE activity. A large family of CaM-PDE isozymes is expressed in mammals, all of which can be activated by Ca2+/CaM. The sequences for the lung 59-kDa, brain 61-kDa, and brain 63-kDa CaM-PDEs have been determined (22-24). Two other forms of 68 kDa and 75 kDa have been extensively purified (25, 26). In addition, a high-affinity CaM-PDE activity enriched in olfactory cilia has been reported (16). These isozymes differ in subunit composition, molecular weight, substrate kinetics, regulatory properties, and localization. We now report the cDNA cloning, expression, and characterization of another CaM-PDE.§ This CaM-PDE has high affinity for cAMP and cGMP and its mRNA is highly enriched in olfactory sensory neurons, indicating that this enzyme may mediate a Ca2+-regulated rapid termination of olfactory cyclic nucleotide signaling, a specific physiological demand for effective olfactory transduction.
ABSTRACT The sensing of an odorant by an animal must be a rapid but transient process, requiring an instant response and also a speedy termination of the signal. Previous biochemical and electrophysiological studies suggest that one or more phosphodiesterases (PDEs) may play an essential role in the rapid termination of the odorant-induced cAMP signal. Here we report the molecular cloning, expression, and characterization of a cDNA from rat olfactory epithelium that encodes a member of the calmodulin-dependent PDE family designated as PDE1C. This enzyme shows high affinity for cAMP and cGMP, having a K. for cAMP much lower than that of any other neuronal Ca2+/calmodulin-dependent PDE. The mRNA encoding this enzyme is highly enriched in olfactory epithelium and is not detected in six other tissues tested. However, RNase protection analyses indicate that other alternative splice variants related to this enzyme are expressed in several other tissues. Within the olfactory epithelium, this enzyme appears to be expressed exclusively in the sensory neurons. The high affinity for cAMP of this Ca2 /calmodulin-dependent PDE and the fact that its mRNA is highly concentrated in olfactory sensory neurons suggest an important role for it in a Ca2+-regulated olfactory signal termination.
Many odorants activate olfactory sensory neurons through guanine nucleotide-binding protein (G protein)-coupled receptors that elicit rapid and transient pulses of intracellular cAMP (1, 2), which directly activates a cyclic nucleotide-gated (CNG) channel allowing an influx of Na+ and Ca2+ ions (3). Many of the components of this transduction pathway have been cloned, including a family of odorant receptors (4), a G protein (Golf) (5), an adenylyl cyclase (6), and CNG channels (7, 8). The transient nature of the olfactory transduction signal is thought to be particularly important for these neurons so that they can be repeatedly stimulated. Following stimulation, cAMP levels can decline nearly as rapidly (within -200 msec) as they can rise (within 50-100 msec) (9). This raises the question of how the odorant-induced second messenger cascade is turned off. Although shutoff of cAMP synthesis may be required and can be achieved by the phosphorylation of odorant receptors by protein kinase A (10, 11), or f3-adrenergic receptor kinase (12, 13), recent studies strongly indicate that the rapid decline of odorant-induced cAMP also must involve hydrolysis of cAMP by one or more phosphodiesterases (PDEs). For example, the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) prolongs the normally transient elevation of cAMP (11) and the current elicited by odorant stimulation (14). In addition, the predominant PDE activity in olfactory cilia is Ca2+/calmodulin dependent and studies using rapid stop-flow analysis indicate that cAMP accumulation in the subsecond, time range is disproportionately enhanced in the presence of IBMX at higher concentrations of Ca2+,
MATERIALS AND METHODS Cloning andi Sequencing of PDE1C2. We screened a rat olfactory cDNA library (kindly provided by F. F. Borisy, University of Chicago) using a probe derived from a PCR fragment of PDE1A. The sequences of the PCR primers were 5'-TTGATGAAACAAGGAGACTGCTGGA-3' (+320 -+344 of bovine PDE1A2) and 5'-TATGCTCATAGTCATGAATGGCAGC-3' (+863 -> +887 of bovine PDE1A2), corresponding to bovine PDE1A2 (61-kDa CaM-PDE) sequences that are conserved between bovine PDE1A2 and PDElB1 (63-kDa CaM-PDE) (22, 23). The PCR was performed at an annealing temperature of 37°C for 30 cycles of the following program: 94°C, 45 sec; 37°C, 1 min; 72°C, 1 min. The resulting 585-nt fragments were subcloned into the TA II vector (Invitrogen). Several colonies that did not hybridize to either a PDE1A2 or a PDElB1 probe were isolated and sequenced. A PCR product with a novel CaM-PDE sequence was used to further screen a rat olfactory cDNA library (27). Abbreviation: PDE, phosphodiesterase; CaM-PDE, Ca2+/calmodulin-dependent phosphodiesterase; CNG, cyclic nucleotide-gated; ISH, in situ hybridization. tTo whom reprint requests should be addressed. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. L41045).
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Filters were washed at high stringency (0.1 x SSC buffer/0.5% SDS at 65°C). About 1000 hybridizing plaques were detected from 1 x 106 recombinants. Twenty were analyzed further and 18 represented PDE1C2. The cDNAs were sequenced on both strands by constructing a series of nested deletions using an Erase-a-Base system from Promega. The longest cDNA clone covers the region from 154 to 3187 (Fig. 1). The first 153 nt were obtained via a 5' RACE system (rapid amplification of cDNA ends, BRL) from rat olfactory first-strand cDNA. RNase Protection. Total RNA was isolated from various rat tissues according to Chomczynski and Sacchi (28). The RNA samples were stored in 100% formamide at a concentration of 2 mg/ml. The P1 and P2 riboprobes were synthesized in vitro from the SP6 or T7 promoters in PDE1C2 subclones. The size of P1 probe is 261 bp (+1 -> +261), and the size of P2 is 300 bp (+ 1783 -* +2083). Rat f-actin riboprobe was prepared from the vector pTRI-,B-Actin-125-Rat (Ambion). The specific activity of the probes was about 109 cpm/,ug. RNase protection analysis was carried out as described by Zhao et al. (29) with slight modifications. For each reaction, we used 12 ,ug of total RNA (25 ,ug for lung and intestine). The probe (P1 or P2) was mixed with the f3-actin control probe in each reaction and allowed to hybridize with the RNA samples at 50°C for 16 hr. kNase digestion was carried out with 40' ,g of RNase A per rhl and 20 units of RNase'Ti per ml at 37°C for 1 hr. The resultant protected RNA fragments were resolved'on a 5% denaturing polyacrylamide/urea gel. In Situ Hybridization (ISH). Unilaterally bulbectomized male rats (150-200 g; Taconic Labs) were anesthetized with pentobarbital and perfused intracardially with 4% paraformaldehyde in phosphate-buffered saline (PBS) 6-8 days after surgery. Rat snouts were then dissected, postfixed overnight in 4% paraformaldehyde in PBS, and then sunk in 15% (wt/vol) sucrose for 24 hr. Tissue blocks were embedded in Tissue-Tek (Miles), stored at -70°C until use, and then sectioned in a cryostat at 16 ,um. To test for specificity, two 35S-labeled antisense riboprobes corresponding to different domains of the same gene were used, including probes from the coding region (748-1329) and the 3' noncoding sequence (24782886) (Fig. 1). The riboprobes derived from different regions gave identical labeling patterns and the signal intensities were roughly proportional to their sizes. Other procedures for ISH were performed as described (30). Transient Expression and Assay of PDE Activity. A 3034nt-long PDE1C2 cDNA (from- 154 to 3187; see Fig. 1) was ligated into EcoRI-digested pCDNA3 vector DNA. The resulting pCDNA3-PDElC2 was used to transfect COS-7 cells with a calcium phosphate transfection kit (Invitrogen). Transfected cells were homogenized in a buffer containing 40 mM Tris HCl (pH 7.5), 5 mM EDTA, 15 mM benzamidine, 15 mM 2-mercaptoethanol, 1 jig of pepstatin A per ml, and 1 jig of leupeptin per ml using a Dounce homogenizer. Homogenates were assayed for PDE activity according to the method of Hansen and Beavo (31) in a buffer containing 20 mM Tris-HCl (pH 7.5), 20 mM imidazole (pH 7.5), 3 mM MgCl2, 15 mM magnesium acetate, 0.2 mg of bovine serum albumin per ml, and [3H]cAMP or [3H]cGMP (100,000 cpm per tube) with either 2 mM EGTA or 0.4 mM CaCl2 and 4 ,ug of CaM per ml. Activity is expressed as nmol of cAMP or cGMP hydrolyzed per min/mg of cellular protein. The protein concentration was estimated according to Bradford (32) using bovine serum albumin as a standard.
RESULTS Using a reverse transcription-PCR approach with primers derived from cDNA sequences that are conserved between the bovine PDE1A2 and PpElBl (22, 23), we obtained a PCR product from mouse brain first-strand cDNA that appeared to represent part of a CaM-PDE gene referred to as PDE1C. A
Proc. Natl. Acad. Sci. USA 92 (1995) very high level of expression was detected in olfactory epithelium for the PDE1C gene but not for PDE1A and PDE1B genes by ISH (data not shown), suggesting that PDE1C might encode the high-affinity CaM-PDE activity previously detected in rat olfactory cilia (16). To isolate cDNAs for this olfactory-enriched CaM-PDE, we screened a rat olfactory cDNA library (27). Approximately 1 in every 1000 recombinant cDNA clones represented this PDE1C, similar to what has been observed for type III adenylyl cyclase in the same library (6). Olfactory adenylyl cyclase is - 100 times more abundant in ciliary membranes than the adenylyl cyclase in myocardial membranes (33), suggesting that the expression level 'of this olfactory CaM-PDE is also very high. Sequence analysis of the longest of these cDNA clones revealed an open reading frame of 2307 nt (Fig. 1). Although a good consensus sequence for initiation of translation was present at the first ATG codon (34), there were no stop codons preceding it. To determine if the first ATG codon was the initiator methionine, we obtained an additional 153 nt of further upstream 5' sequence by a 5' RACE system and found stop codons in all three reading frames. Thus, the first methionine is likely to be the initiator for a cDNA encoding a 768-amino acid polypeptide having a calculated molecular mass of 86.67 kDa. The tissue distribution of PDE1C mRNA was examined by RNase protection assay (Fig. 2). Many PDE genes can be expressed in multiple splice forms that often differ at their 5' ends (35). Therefore, we used two different probes: P1, which corresponds to 5' noncoding sequence, and P2, which corresponds to a region in the catalytic domain (Fig. 2A). The P2 probe detects a high level of PDE1C expression in olfactory epithelium and moderate expression in cerebellum as well as weak expression in forebrain, testis, heart, and lung (Fig. 2C). The P1 probe is fully protected only in olfactory epithelium (Fig. 2B). The strong signal in the f3-actin control indicates that the RNA samples were not significantly degraded (Fig. 2 B and C). These observations are most consistent with the idea that the rat PDE1C cDNA is a splice variant of the PDE1C gene that is expressed in olfactory epithelium. We' designate this particular splice variant as PDE1C2 since another full-length PDE1C cDNA recently isolated from human and mouse tissues is called PDElC1 (refs. 36 and 37; C.Y., A.Z.Z., J.K.B., and J.A.B., unpublished observation). The data also suggest that additional, alternative splice variants are expressed in other rat tissues. The localization of PDE1C mRNA within the olfactory epithelium was examined by ISH using a neuronal depletion technique. Sensory neurons but not epithelial cells of the olfactory epithelium degenerate 6-8 days after the removal of the neuronal target tissue, the olfactory bulb. Removal of one side of the olfactory bulb leads to ipsilateral degeneration of the receptor neurons within the olfactory epithelium. The very intense signal seen in the intact side was greatly reduced on the bulbectomized side (Fig. 3). The disappearance of'PDElC mRNA concomitant with the loss of sensory neurons after bulbectomy strongly indicates that PDEtC 'is expressed in olfactory sensory neurons. A particularly high level of PDE1C gene expression in olfactory sensory neurons was indicated by the observation that the ISH signal could be detected in
