Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor.

Abstract

There is increasing evidence that pituitary ATP receptors may play a novel role in modulating pituitary function. This work reports the isolation and expression of a pituitary ATP receptor gene clone from a rat pituitary complementary DNA library. The isolated clone (rpP2U) has a 1125-bp coding sequence flanked by 483 bp of 5' - and 422 bp of 3'-untranslated sequences. The deduced 374-amino acid product shows structural features common to other G protein-coupled receptors, and when stably transfected into a glioma cell line lacking endogenous ATP receptors, is functionally characterized as a P2U purinoceptor. Specifically, the ATP-induced intracellular Ca2+ mobilization in the transfected cells was inhibited by suramin, 2-methylthio-ATP had a modest stimulatory effect on intracellular Ca2+ mobilization, and beta, gamma-methylene ATP and alpha, beta-methylene ATP had no effect. The cloned receptor exhibited the agonist potency and efficacy profile of ATP approximately equal to uridine triphosphate > ADP approximately equal to uridine diphosphate > GTP. Such characteristics very closely mimic the pharmacologically defined P2U purinoceptor of primary rat gonadotropes and mixed sheep pituitary cells, and Southern blot analysis further indicates that there is only one allele in rat genome for the P2U purinoceptor. These findings suggest that the P2U purinoceptor is the predominant G protein-linked ATP receptor found in the pituitary.

Expression of P2Y receptors in the rat anterior pituitary

“…UTP-sensitive receptors have been suggested to be present in gonadotrophs [16,26] and lactotrophs [17], but these results were not confirmed by other reports [2,14]. The present results showed that gonadotrophs and lactotrophs express P2Y 4 and P2Y 2 receptors, respectively, the ligand for which is UTP, confirming that gonadotrophs and lactotrophs express UTP-sensitive receptors.…”

“…Following RT-PCR analysis, P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 12 receptor transcripts were detected in mixed anterior pituitary cells [13,14]. The identified cells in the anterior pituitary with P2Y receptors were also found in rat pituitary folliculo-stellate cells in primary culture [15], gonadotrophs [16] and lactotrophs [17]. No detailed immunofluorescence, Western blot or in situ hybridization studies about P2Y receptor subtype expression on the individual cell types in the anterior pituitary are currently available.…”

Multiple P2 Receptors Contribute to a Transient Increase in Intracellular Ca2+ Concentration in Atp‐Stimulated Rat Brown Adipocytes

“…The sequencing analysis revealed that the PCR product of this major band was an alternative splice variant lacking 72 bp (nt 339-410 in GenBank accession number X97328). In metabotropic P2Y receptor subtypes, amplification of P2Y 1 , P2Y 2 , P2Y 4 and P2Y 6 receptor subtypes genes resulted in single bands around the predicted sizes of 415, 359, 352 and 313 bp, respectively, and PCR products were found to show 100 % homology for the sequences reported previously (Chang et al 1995;Tokuyama et al 1995;Chen et al 1996;Bogdanov et al 1998). The expression of P2Y 11 receptor subtype gene was not detected by using human P2Y 11 primers in these rat brown adipocytes.…”

“…Single strand cDNA was synthesized from 5 mg of total RNA with 20 units of RAV-2 reverse transcriptase (Takara Biochemicals, Kyoto, Japan) using random primers. A portion of cDNA was amplified by the polymerase chain reaction (PCR) method using rat P2X 1 -, P2X 2 , P2X 3 -, P2X 4 -, P2X 5 -, P2X 6 -, P2X 7 -, P2Y 1 -, P2Y 2 -, P2Y 4 -, P2Y 6 -and human P2Y 11 -specific primers synthesized according to the nucleotide sequences of rat receptor genes P2X 1 (GenBank accession number X80477) (Valera et al 1994), P2X 2 (GenBank accession number U14414) (Brake et al 1994), P2X 3 (GenBank accession number X90651) (Chen et al 1995), P2X 4 (GenBank accession number U47031) (Wang et al 1996), P2X 5 (GenBank accession number X97328) (Garcia-Guzman et al 1996), P2X 6 (GenBank accession number X92070) (Collo et al 1995), P2X 7 (GenBank accession number X95882) (Surprenant et al 1996), P2Y 1 (GenBank accession number U22830) (Tokuyama et al 1995), P2Y 2 (GenBank accession number L46865) (Chen et al 1996), P2Y 4 (GenBank accession number Y14705) (Bogdanov et al 1998), P2Y 6 (GenBank accession number D63665) (Chang et al 1995) and P2Y 11 (GenBank accession number AF030335) (Communi et al 1997), respectively: P2X 1 sense, 5‚-CCC-AGAAGGTGGCATATGCCAG-3‚ (nt 569-590); P2X 1 antisense, 5‚-CAGTCCAGATCACACTTCCAGTC-3‚ (nt 1003-981); P2X 2 sense, 5‚-ATCCTCATCAAGAACAGCATCCAC-3‚ (nt 589-612); P2X 2 antisense, 5‚-TTGGCAAACCTGAAGTTGTAGCCT-3‚ (nt 914-891); P2X 3 sense, 5‚-CTGTGAGATCCAGGGCTGGTG-3‚ (nt 619-639); P2X 3 antisense, 5‚-GGATGCCAAAAGCCT-TCAGGAG-3‚ (nt 1091-1070); P2X 4 sense, 5‚-AACATGATT-GTCACCGTGAACCAG-3‚ (nt 340-363); P2X 4 antisense, 5‚-TGCCATCACCTGGAAGCTATGTC-3‚ (nt 771-749); P2X 5 sense, 5‚-GACACTTCCCTGCAGAGTGCTG-3‚ (nt 225-246); P2X 5 antisense, 5‚-AAGGGGATCCGTTGGCATGGAC-3‚ (nt 593-572); P2X 6 sense, 5‚-GTAACCCAGGTTAAGGAACTG-GAG-3‚ (nt 239-262); P2X 6 antisense, 5‚-GTATCTAAG-GCATTGGTTCTGGAG-3‚ (nt 622-645); P2X 7 sense, 5‚-TCT-TCGACACGGCCGACTACAC-3‚ (nt 381-402); P2X 7 antisense, 5‚-AGCCGGAAGATGGGACACTGAG-3‚ (nt 813-792); P2Y 1 sense, 5‚-TCCGATGTGCCCTGATCAAGAC-3‚ (nt 738-759); P2Y 1 antisense, 5‚-ATGAGCCATACCAGCACACTGAC-3‚ (nt 1152-1130); P2Y 2 sense, 5‚-CGCTTCAACGAGGACTTCAAG-TA-3‚ (nt 559-581); P2Y 2 antisense, 5‚-CCCCAGCTCAGG-GAGTGCAGA-3‚ (nt 917-897); P2Y 4 sense, 5‚-GTTCTG-GAGATGGTGATTGTAGG-3‚ (nt 1407-1429); P2Y 4 antisense, 5‚-CAGTATCCCAGGTATCGGTGCA-3‚ (nt 1758-1737); P2Y 6 sense, 5‚-CTACCGTGAGGATTTCAAGCGAC-3‚ (nt 496-518); P2Y 6 antisense, 5‚-CTGGAAGCTAATGCAGGTGAGGA-3‚ (nt 808-786); P2Y 11 sense, 5‚-TGGAGCGCTTCCTCTTCACCTG-3‚ (nt 302-323); P2Y 11 antisense, 5‚-AGTCATGCCTGGGCT-GCGTAG-3‚ (nt 708-688). The PCR reaction included 0.4 ml of template cDNA, 2.5 units of KOD dash polymerase (Toyobo Biochemicals, Osaka, Japan), 1 mM KCl, 6 mM (NH 4 ) 2 SO 4 , 0.1 % Triton X-100, 10 mg ml _1 BSA, 0.2 mM each of deoxynucleotide triphosphates and 4 pmol primers in 20 ml of 120 mM Tris-HCl buffer (pH 8.0).…”

Therapeutic potential of extracellular ATP and P2 receptors in nervous system diseases

“…P2Y 1 is a receptor for the endogenous ligands ADP, more than ATP and diadenosine polyphosphates. The P2Y 2,4 receptors are activated with approximately equal potency by ATP as well as UTP [11] . P2Y 6 , showing preference for UDP, is a receptor for pyrimidine which is activated specifically by uridine nucleotides and not by adenine nucleosides or nucleotides [12] .…”