Cheung P P, Xu H, McLaughlin M M, Ghazaleh F A, Livi G P, Colman R W
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Blood. 1996 Aug 15;88(4):1321-9.
Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists. Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP. The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate [cGMP]-inhibited PDE). To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins. This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence. The nucleotide coding for the N terminal 179 amino acid sequence (nt 1-536) as well as four other cDNAs (nt 1459-1632, nt 1765-1986, nt 2152-2538, and nt 2978-3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same. Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb. Four new deletion mutants are reported. PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast. They displayed PDE activities of 172 and 79 pmol/mg/min, respectively. PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity. All mutant proteins were expressed as determined by Western blot analysis. These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.
环磷酸腺苷（cAMP）是血小板对激动剂反应的重要调节因子。环核苷酸磷酸二酯酶（PDE）通过将cAMP水解为AMP来控制细胞内cAMP浓度。血小板中的主要PDE活性是PDE3A（环鸟苷酸[cGMP]抑制性PDE）。为了获得血小板PDE3A的结构信息，我们从人红白血病细胞（HEL）文库中克隆了该酶的cDNA，因为该细胞系表达许多血小板蛋白。该克隆包含全长人心肌PDE3A cDNA的87%，跨度为核苷酸456至4606，且序列相同。通过血小板RNA的逆转录聚合酶链反应（RT-PCR）获得的编码N端179个氨基酸序列（核苷酸1-536）的核苷酸以及其他四个cDNA（核苷酸1459-1632、核苷酸1765-1986、核苷酸2152-2538和核苷酸2978-3375）也与心肌序列相同，表明HEL、心肌和血小板PDE3A是相同的。对HEL细胞RNA的Northern印迹分析检测到7.5 kb和4.4 kb的两种mRNA。报告了四个新的缺失突变体。分别编码氨基酸665至1141和氨基酸679至1141的PDE 3Aδ1和PDE 3Aδ2在缺乏PDE的酵母中表达。它们的PDE活性分别为172和79 pmol/mg/分钟。编码氨基酸686至1141和700至1141的PDE 3Aδ3和PDE 3Aδ4没有可检测到的PDE活性。通过蛋白质免疫印迹分析确定所有突变蛋白均已表达。这些发现将PDE3A催化域定位在氨基酸残基679至1141内。
