G proteins mediate the interaction between cell surface receptors and intracellular effectors. Recent studies have shown that human retina and rat brain contain mRNA encoding a novel 40-Kd G protein α subunit referred to as G(zα). Studies with an antiserum selective for the predicted sequence of this protein have suggested that a similar protein is present in human platelets and is phosphorylated during platelet activation. To better understand the structure and function of this protein, the present studies examine its sequence in platelets and compare its abundance in human platelets, megakaryocytes, and two megakaryoblastic cell lines, HEL cells and Dami cells. Three different G(zα)-selective antisera reacted with a 40-Kd protein in platelet membranes. None of these detected a corresponding protein in HEL or Dami cells, despite the presence in both cell lines of proteins recognized by antisera selective for three members of the G(iα) family. Northern blotting with a G(zα)-specific probe prepared from retinal G(zα) showed two hybridizing species in platelet RNA: a major band at 3.5 kb and a minor band at 2.2 kb. Both were detectable in HEL and Dami cells, but at greatly reduced levels compared with platelets. RNA encoding G(zα) was also detected in individual human megakaryocytes by in situ hybridization. The amount present approached that of G(iα2), the most abundant of the G(iα) species present in platelets. The complete sequence of the platelet homolog to G(zα) was determined from platelet RNA amplified by the polymerase chain reaction. The encoded protein was the same as those obtained in brain and retina. Thus, based on immunoreactivity and nucleotide sequencing, platelets and megakaryocytes contain substantial quantities of a protein identical to brain and retinal G(zα). The paucity of G(zα) protein and RNA in the megakaryoblastic cell lines suggests that either there has been a selective loss of the ability to synthesize G(zα) from these cells or that G(zα) appears at a later stage in megakaryocyte development than does G(iα).
Gagnon A.W., Manning D.R., Catani L., Gewirtz A., Poncz M., Brass L.F. (1991). Identification of G(zα) as a pertussis toxin-insensitive G protein in human platelets and megakaryocytes. BLOOD, 78(5), 1247-1253 [10.1182/blood.v78.5.1247.1247].
Identification of G(zα) as a pertussis toxin-insensitive G protein in human platelets and megakaryocytes
Catani L.;
1991
Abstract
G proteins mediate the interaction between cell surface receptors and intracellular effectors. Recent studies have shown that human retina and rat brain contain mRNA encoding a novel 40-Kd G protein α subunit referred to as G(zα). Studies with an antiserum selective for the predicted sequence of this protein have suggested that a similar protein is present in human platelets and is phosphorylated during platelet activation. To better understand the structure and function of this protein, the present studies examine its sequence in platelets and compare its abundance in human platelets, megakaryocytes, and two megakaryoblastic cell lines, HEL cells and Dami cells. Three different G(zα)-selective antisera reacted with a 40-Kd protein in platelet membranes. None of these detected a corresponding protein in HEL or Dami cells, despite the presence in both cell lines of proteins recognized by antisera selective for three members of the G(iα) family. Northern blotting with a G(zα)-specific probe prepared from retinal G(zα) showed two hybridizing species in platelet RNA: a major band at 3.5 kb and a minor band at 2.2 kb. Both were detectable in HEL and Dami cells, but at greatly reduced levels compared with platelets. RNA encoding G(zα) was also detected in individual human megakaryocytes by in situ hybridization. The amount present approached that of G(iα2), the most abundant of the G(iα) species present in platelets. The complete sequence of the platelet homolog to G(zα) was determined from platelet RNA amplified by the polymerase chain reaction. The encoded protein was the same as those obtained in brain and retina. Thus, based on immunoreactivity and nucleotide sequencing, platelets and megakaryocytes contain substantial quantities of a protein identical to brain and retinal G(zα). The paucity of G(zα) protein and RNA in the megakaryoblastic cell lines suggests that either there has been a selective loss of the ability to synthesize G(zα) from these cells or that G(zα) appears at a later stage in megakaryocyte development than does G(iα).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.