Gα16, a G Protein α Subunit Specifically Expressed in Hematopoietic Cells

Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein α subunit, Gα16, is specifically expressed in human cells of the hematopoietic lineage. The Gα16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the Gq class of α subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein α subunits, the Gα16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the Gα16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. Gα16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of Gα16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress Gα16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that Gα16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin

Distinct forms of the ß subunit of GTP-binding regulatory proteins identified by molecular cloning

Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 2 subunits. The bovine transducin β subunit (β 1) has been cloned previously. We have now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue Mr 37,329 β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expreβed at lower levels than β 1 subunit mRNA in all tiβues examined. The β 1 and β 2 meβages are expreβed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

Specific Interactions of Chemoattractant Factor Receptors with G-proteins

Stimulation of leukocytes with chemoattractant ligands activates phospholipid turnover and calcium release, ultimately leading to chemotaxis, degranulation, and the inflammatory response. The leukocyte response to these ligands is transduced by the interaction of transmembrane receptors with GTP-binding regulatory proteins (G-proteins). To examine the mechanisms of signal transduction by these receptors, we transfected cDNA clones encoding the receptors for the active cleavage product of the fifth component of complement (C5a) and platelet-activating factor (PAF) into COS-7 cells, then measured the production of inositol phosphates (IP) in response to stimulation with these chemoattractant ligands. Cells transfected with the C5a receptor showed no increase in IP production when stimulated with ligand (5-120 nM). However, in cells co-transfected with these receptors and with the cDNA for Gα_(16), a G-protein α subunit that is specific to cells of hematopoietic lineage, addition of ligand caused up to a 5-fold increase in IP production. This interaction was specific, as co-transfection of receptors with the G-proteins Gα_q or Gα_(11) did not allow ligand-dependent increase in IP production. In contrast, ligand-dependent activation of IP production was seen in COS cells transfected solely with the PAF receptor. These results indicate that the C5a receptor utilizes signaling pathways distinct from the PAF receptor and suggest that a pertussis toxin-resistant G-protein, Gα_(16), may play a role in the leukocyte response to inflammatory ligands

The 35- and 36-kDa β Subunits of GTP-binding Regulatory Proteins Are Products of Separate Genes

The wide range of functions attributed to GTP-binding regulatory proteins (G proteins) is reflected in the structural diversity which exists among the α, β, and y subunits of G proteins. Recently two cDNA clones encoding β subunits, β_1 and β_2, were isolated from bovine and human cDNA libraries. We report here that the β_2 gene encodes the 35-kilodalton (kDa) component of the β_(35)/β_(36) subunit of G proteins and that the β_1 gene encodes the 36-kilodalton component. The in vitro translation product of the β_2 cDNA co-migrates with the 35-kDa β subunit (β_(35)), while the in vitro product of the β_1 cDNA co-migrates with the 36-kDa β subunit (β_(36)) on denaturing polyacrylamide gels. In addition, antisera generated against synthetic β_2 peptides bind specifically to the β_(35) component of isolated G proteins and to a 35-kDa protein in myeloid cell membranes. Our results suggest that the two β subunits could serve distinct functions, as they are derived from separate genes which have been highly conserved in evolution

Evolution of the mammalian G protein α subunit multigene family

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. The multigene family of G protein α subunits, which interact with receptors and effectors, exhibit a high level of sequence diversity. In mammals, 15 Gα subunit genes can be grouped by sequence and functional similarities into four classes. We have determined the murine chromosomal locations of all 15 Gα subunit genes using an interspecific backcross derived from crosses of C57BI/6J and Mus spretus mice. These data, in combination with mapping studies in humans, have provided insight into the events responsible for generating the genetic diversity found in the mammalian α subunit genes and a framework for elucidating the role of the Gα subunits in disease