Identification of the Pertussis Toxin-Sensitive G Proteins in Platelets, Megakaryocytes, and Human Erythroleukemia Cells

• Published in: Blood Vol. 76; no. 4; pp. 721 - 730
• Main Authors: Williams, Alison G., Woolkalis, Marilyn J., Poncz, Mortimer, Manning, David R., Gewirtz, Alan M., Brass, Lawrence F.
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Washington, DC Elsevier Inc 15.08.1990; The Americain Society of Hematology
• Subjects: Adenylate Cyclase Toxin; Amino Acid Sequence; Antibodies - immunology; Base Sequence; Biological and medical sciences; Blood coagulation. Blood cells; Blood Platelets - metabolism; DNA - genetics; Electrophoresis, Gel, Two-Dimensional; Fundamental and applied biological sciences. Psychology; GTP-Binding Proteins - genetics; GTP-Binding Proteins - immunology; GTP-Binding Proteins - metabolism; Humans; Immunoblotting; Leukemia, Erythroblastic, Acute - blood; Leukemia, Erythroblastic, Acute - metabolism; Leukemia, Erythroblastic, Acute - pathology; Megakaryocytes - metabolism; Molecular and cellular biology; Molecular Sequence Data; Nucleic Acid Hybridization; Pertussis Toxin; Platelet; Precipitin Tests; Virulence Factors, Bordetella - pharmacology
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V76.4.721.721

Guanine nucleotide-binding regulatory proteins, or G proteins, mediate the interaction of agonist receptors on the platelet surface with phospholipase C and adenylyl cyclase. To better understand this process, we have used several approaches to identify which G proteins are present in platelets, normal human megakaryocytes, and human erythroleukemia (HEL) cells, a leukemic cell line with megakaryocytic features. Because platelet and HEL cell responses to thrombin are inhibited by pertussis toxin, we have focused upon the members of the Gi family, whose α subunits can be ADP-ribosylated by that toxin. Western blots with antisera specific for Giα demonstrated the presence in both platelets and HEL cells of the three best-described forms of this protein: Giα1, Giα2, and Giα3. Based upon immunoprecipitation studies with [35S]-methionine-labeled HEL cells, their relative abundance appears to be Giα2 > Giα3 > Giα1. A HEL cell cDNA library screened with the Giα antisera produced clones encoding Giα2 and Giα3 that had sequences similar to those reported from other sources. Giα-specific probes created from these cDNA clones confirmed the presence of mRNA encoding Giα2 and Giα3 in both platelets (by Northern blotting) and megakaryocytes (by in situ hybridization). Thus the pertussis toxin substrates that have previously been detected in platelets and HEL cells are shown to be members of the Giα family, all of which are candidates for interaction with receptors for thrombin and other agonists.