Detection of multiple forms of G_iα in HL60 cells

Abstract

Comparison of cDNA sequences from multiple sources predicts a genus of highly homologous but structurally distinct G protein α‐subunits, designated as Giα, that may include the α‐subunit of the functionally defined adenylate cyclase inhibitory G protein. Using specific oligonucleotide probes on Northern blots, we show that Giα−2 and Giα−3, but not Giα−1, are expressed in HL60 cells. Antisera raised against synthetic peptides from regions predicted to be conserved (AS6) and divergent (LE3) among Giα subtypes bind to a 40 kDa protein in Western blots of HL60 membranes. AS6 identifies an additional protein at 41 kDa. Thus, Northern blot and immunoblot results show that at least two Giα subtypes, a 40 kDa protein likely to correspond to Giα−2 and a 41 kDa protein possibly representing Giα−3, may be expressed in a single cell type.

Cloning and characterization of the human gene for the α‐subunit of Gi2, a GTP‐binding signal transduction protein

“…850 X&I to SphI genomic fragment from A42 which is just downstream of the region homologous to the 3 ' end of isolated cDNAs [4,17] the approx. 0.5 kb discrepancy between reported cDNA length (about 1.7 kb) [4,17,18] and Giza mRNA size (about 2.2 kb) as seen on RNA hybridization analys,es by us and others [3,4,7,18]. It is likely that during cDNA synthesis, oligo(dT) priming at the A-rich region instead of the poly(A) tail resulted in cDNAs incomplete at the 3 ' end by approx.…”

“…nylon filters [7]. DNA fragments were labelled by random priming [lo] and filters were hybridized and washed as described [3].…”

“…RNA blot hybridization analysis of HL60 RNA with genomic and cDNA probes. HL60 cells were induced to maturation with dibutyryl-CAMP and RNA isolated [7]. 15 pg unselected HL60 RNA (lanes 1 and 3) were size separated on an agarose gel adjacent to ribosomal RNA, 5 gg (lanes 2 and 4), and then transferred to nylon (Nytran) filters.…”

“…Giz may act in neutrophils to couple chemotactic factor receptors to stimulation of phospholipase C [6]. Expression of Giz, measured by protein and RNA analyses, is greatly enhanced when HL-60 and U-937 cells are induced to differentiate by agents such as dibutyryl-CAMP and dimethylsulfoxide [7]. In order to examine the regulation of the Gi2 gene, and to define the exon structure of a G-protein a-subunit gene, we have isolated and characterized human genomic DNA clones encompassing the GiZa subunit gene and its 5 ' and 3 ' flanking regions.…”

Nucleotide-, Chemotactic Peptide- and Phorbol Ester-Induced Exocytosis in HL-60 Leukemic Cells

“…Autoradiography of dried polyacrylamide gels was performed as described (25). Gels were cut on the basis of autoradiographs, and radioactivity incorporated into gel bands corresponding to 40-41 kDa proteins, presumably representing the a-subunits of Gl2 and Gi3 (38), was determined by liquid scintillation counting. Table 2).…”