Human Platelet Gαq Deficiency Is Associated with Decreased Gαq Gene Expression in Platelets but not Neutrophils^[*]

Summary

G-proteins play an important role in platelet signal transduction and regulate responses upon activation of G-protein coupled receptors (GPCR). We have previously reported a patient with impaired platelet responses associated with deficiency in platelet Gαq. To understand the molecular basis for this defect, the cDNA sequence encoding Gαq (1080 bp) was obtained by reverse-transcription and polymerase chain reaction of platelet RNA; the cDNA sequence showed no mutations in the patient. Platelet Gαq mRNA levels were decreased by >50% compared to normal subjects; platelet Gαi2 mRNA levels were normal. Neutrophil calcium mobilization and elastase secretion, upon activation with several agonists, and neutrophil Gαq mRNA and protein levels were normal. These studies demonstrate that the patient has a defect in Gαq gene expression in platelets but not neutrophils, possibly due to defects in transcriptional regulation or mRNA stability, and suggest a hematopoietic-lineage specific defect.

Keywords

Platelet activation - Gαq - G-proteins - gene expression - signal transduction

^* Supported by R01HL 56724 from the National Heart, Lung and Blood Institute (AKR)

• References

• 1 Brass LF. More pieces of the platelet activation puzzle slide into place. J Clin Invest 1999; 104: 1663-5.
• 2 Hamm HE. The many faces of G protein signaling. J Biol Chem 1998; 273: 669-72.
• 3 Farfel Z, Bourne HR, Iiri T. The expanding spectrum of G protein diseases. N Engl J Med 1999; 340: 1012-20.
• 4 Dhanasekaran N, Heasley LE, Johnson GL. G-protein-coupled receptor systems involved in cell growth and oncogenesis. Endocrine Review 1995; 16: 259-70.
• 5 Spiegel AM. G protein defects in signal transduction. Horm Res 2000; 53: 17-22.
• 6 Rao AK, Koike K, Willis J. et al. Platelet secretion defect associated with impaired liberation of arachidonic acid and normal myosin light chain phosphorylation. Blood 1984; 64: 914-21.
• 7 Rao AK, Disa J, Yang X. Concomitant defect in internal release and influx of calcium in patients with congenital platelet dysfunction and impaired agonist-induced calcium mobilization: thromboxane production is not required for internal release of calcium. J Lab Clin Med 1993; 121: 52-63.
• 8 Gabbeta J, Yang X, Kowalska MA, Sun L, Dhanasekaran N, Rao AK. Platelet signal transduction defect with Ga subunit dysfunction and diminished Gαq in a patient with abnormal platelet responses. Proc Natl Acad Sci U S A 1997; 94: 8750-5.
• 9 Gabbeta J, Dhanasekaran N, Rao AK. Gαq cDNA sequence from human platelets. Thromb Res 1998; 91: 29-32.
• 10 Chen B, Leverette RD, Schwinn DA, Kwatra MM. Human Gαq: cDNA and tissue distribution. Biochim Biophys Acta 1996; 1281: 125-8.
• 11 Itoh H, Toyama R, Kozasa T, Tsukamoto T, Matsuoka M, Kaziro Y. Presence of three distinct molecular species of Gi protein alpha subunit. Structure of rat cDNAs and human genomic DNAs. J Biol Chem 1988; 263: 6656-64.
• 12 Lounsbury KM, Schlegel B, Poncz M, Brass LF, Manning DR. Analysis of Gz alpha by site-directed mutagenesis. Sites and specificity of protein kinase C-dependent phosphorylation. J Biol Chem 1993; 268: 3494-8.
• 13 Clark RA, Nauseef WM. Isolation and functional analysis of neutrophils. In: Current Protocols in Immunology. Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W. eds. John Wiley and Sons; 1996. 7.23.1-7.23.17..
• 14 Pollock WK, Rink TJ, Irvine RF. Liberation of [³H]arachidonic acid and changes in cytosolic free calcium in fura-2 loaded human platelets stimulated by ionomycin and collagen. Biochem J 1986; 235: 869-77.
• 15 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-50.
• 16 Rao AK, Kowalska MA, Disa J. Impaired cytoplasmic ionized calcium mobilization in inherited platelet secretion defects. Blood 1989; 74: 664-72.
• 17 Haribabu B, Zhelev DV, Pridgen BC, Richardson RM, Ali H, Snyderman R. Chemoattractant receptors activate distinct pathways for chemotaxis and secretion. Role of G-protein usage. J Biol Chem 1999; 274: 37087-92.
• 18 Amatruda TT, Gerard NP, Gerard C, Simon MI. Specific interactions of chemoattractant factor receptors with G- proteins. J Biol Chem 1993; 268: 10139-44.
• 19 Amatruda TT, Steele DA, Slepak VZ, Simon MI. Gα16, a G protein α subunit specifically expressed in hematopoietic cells. Proc Natl Acad Sci USA 1991; 88: 5587-91.
• 20 Jiang H, Kuang Y, Wu Y, Smrcka A, Simon MI, Wu D. Pertussis toxinsensitive activation of phospholipase C by the C5a and fMet-Leu-Phe receptors. J Biol Chem 1996; 271: 13430-4.
• 21 French DL. The molecular genetics of Glanzmann’s thrombasthenia. Platelets 1998; 09: 5-20.
• 22 George JN, Nurden AT. Inherited disorders of the platelet membrane: Glanzmann thrombasthenia, Bernard-Soulier syndrome, and other disorders. In: Hemostasis and Thrombosis. Basic Principles and Clinical Practice. Colman RW, Hirsh J, Marder VJ, Clowes A, George JN. eds. Philadelphia: J.B. Lippincott; 2001: 921-43.
• 23 Carey M, Smale S. Modes of regulating mRNA abundance. Transcriptional Regulation in Eukaryocytes. New York: Cold Spring Harbor Laboratory Press; 2000: 65-97.
• 24 Lee WMF, Dang CV. Control of cell growth and differentiation. In: Hematology: Basic Principles and Practice. Hoffman R, Benz Jr EJ, Shattil S, et al. eds. 3rd ed. Philadelphia: Churchill Livingstone; 2000: 57-71.
• 25 Shivdasani RA, Orkin SH. The transcriptional control of hematopoiesis. Blood 1996; 87: 4025-39.
• 26 Uzan G, Prandini MH, Berthier R. Regulation of gene transcription during the differentiation of megakaryocytes. Thromb Haemost 1995; 74: 210-2.
• 27 Prandini MH, Martin F, Thevenon D, Uzan G. The tissue-specific transcriptional regulation of the megakaryocytic glycoprotein IIb gene is controlled by interactions between a repressor and positive cis-acting elements. Blood 1996; 88: 2062-70.
• 28 Block KL, Shou Y, Poncz M. An Ets/Sp1 interaction in the 5’-flanking region of the megakaryocyte- specific alpha IIb gene appears to stabilize Sp1 binding and is essential for expression of this TATA-less gene. Blood 1996; 88: 2071-80.
• 29 Jin Y, Wilhide CC, Dang C. et al. Human integrin β3 gene expression: evidence for a megakaryocytic cell-specific cis-acting element. Blood 1998; 92: 2777-90.
• 30 Shiraga M, Ritchie A, Aidoudi S. et al. Primary megakaryocytes reveal a role for transcription factor NF-E2 in integrin α IIbβ3 signaling. J Cell Biol 1999; 147: 1419-30.
• 31 Lecine P, Italiano Jr JE, Kim SW, Villeval JL, Shivdasani RA. Hematopoietic-specific β1 tubulin participates in a pathway of platelet biogenesis dependent on the transcription factor NF-E2. Blood 2000; 96: 1366-73.
• 32 Song WJ, Sullivan MG, Legare RD. et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia [see comments]. Nat Genet 1999; 23: 166-75.
• 33 Rao AK, Poncz M. Defective platelet secretory mechanisms in familial thrombocytopenia with transcription factor CBFA2 haplodeficiency. Abstracts of the International Society of Thrombosis and Haemostasis. Thromb Hemost. 2001
• 34 Ludlow LB, Schick BP, Budarf ML. et al. Identification of a mutation in a GATA binding site of the platelet glycoprotein Ibb promoter resulting in the Bernard-Soulier syndrome. J Biol Chem 1996; 271: 22076-80.
• 35 Carew JA, Pollak ES, High KA, Bauer KA. Severe factor VII deficiency due to a mutation disrupting an Sp1 binding site in the factor VII promoter. Blood 1998; 92: 1639-45.
• 36 Arbini AA, Pollak ES, Bayleran JK, High KA, Bauer KA. Severe factor VII deficiency due to a mutation disrupting a hepatocyte nuclear factor 4 binding site in the factor VII promoter. Blood 1997; 89: 176-82.
• 37 Li Y, Mortensen R, Neer EJ. Regulation of ao expression by the 5’-flanking region of the ao gene. J Biol Chem 1994; 269: 27589-94.
• 38 Kinane TB, Shang C, Finder JD, Ercolani L. cAMP regulates G-protein alpha i-2 subunit gene transcription in polarized LLC-PK1 cells by induction of a CCAAT box nuclear binding factor. J Biol Chem 1993; 268: 24669-76.
• 39 Holtzman EJ, Kinane TB, West K. et al. Transcriptional regulation of G-protein ai subunit genes in LLC- PK1 renal cells and characterization of the porcine Gα1-3 gene promoter. J Biol Chem 1993; 268: 3964-75.