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The high‐affinity receptors for human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin 3 (IL‐3) and interleukin 5 (IL‐5) are composed of two distinct subunits, alpha and beta c. The alpha subunits are specific for each cytokine, whereas the beta subunit (beta c) is shared by the three receptors and is an essential component of signal transduction. We have made a series of mutant beta c cDNAs that delete various regions of the cytoplasmic domain and examined the function of these mutants by coexpressing them with the alpha subunit of the human GM‐CSF receptor (hGMR) in an IL‐3‐dependent mouse pro‐B cell line BaF3. Two domains in the membrane‐proximal portion of beta c were found to be important for transducing the hGM‐CSF‐mediated growth signals: one domain between Arg456 and Phe487 appears to be essential for proliferation, and the second domain between Val518 and Asp544 enhances the response to GM‐CSF, but is not absolutely required for proliferation. The region between Val518 and Leu626 was responsible for major tyrosine phosphorylation of 95 and 60 kDa proteins. Thus, beta c‐mediated major tyrosine phosphorylation of these proteins was apparently separated from proliferation. However, the beta 517 mutant lacking residues downstream of Val518 transmitted a herbimycin‐sensitive proliferation signal, suggesting that beta 517 still activates a tyrosine kinase(s). We also evaluated the role of the cytoplasmic domain of the GMR alpha subunit and the results suggest that it is involved in the hGM‐CSF‐mediated signal transduction, but is not essential.(ABSTRACT TRUNCATED AT 250 WORDS)

In a previous paper, we demonstrated that a gene coding for a protein homologous to the alpha subunit of mammalian guanine nucleotide-binding regulatory (G) proteins occurs in Saccharomyces cerevisiae. The gene, designated GPA1, encodes a protein (GP1 alpha) of 472 amino acids with a calculated Mr of 54,075. Here we report the isolation of another G-protein-homologous gene, GPA2, which encodes an amino acid sequence of 449 amino acid residues with a Mr of 50,516. The predicted primary structure of the GPA2-encoded protein (GP2 alpha) is homologous to mammalian G proteins [inhibitory and stimulatory G proteins (Gi and Gs, respectively), a G protein of unknown function (Go), and transducins (Gt)] as well as yeast GP1 alpha. When aligned with the alpha subunit of Gi (Gi alpha) to obtain maximal homology, GP2 alpha was found to contain a stretch of 83 additional amino acid residues near the NH2 terminus. The gene was mapped in chromosome V, close to the centromere. Haploid cells carrying a disrupted GPA2 gene are viable. Cells carrying a high copy number of plasmid GPA2 (YEpGPA2) had markedly elevated levels of cAMP and could suppress a temperature-sensitive mutation of RAS2. These results suggest that GPA2 may be involved in the regulation of cAMP levels in S. cerevisiae.

The GPAI gene of Saccharomyces cerevisiae encodes a protein that is highly homologous to the a subunit of mammalian hetrotrimeric G proteins and is essential for haploid cell growth. A mutation of the GPA1 protein, GPA1Val-50, in which Gly-50 was replaced by valine, could complement the growth defect of a GPAI disruption, gpal::HIS3. However, cells with gpal ::HIS3 expressing the GPAlVaI-S5 protein were supersensitive to a-factor in a short-term incubation but resumed growth after long-term incubation even after exposure to high concentrations of a-factor. The former phenotype associated with GPAJVal-S0 is recessive, and the latter phenotype is dominant to GPAI'. The supersensitivity of GPAVal-S0 to a-factor was dependent on STE2 and STE4, which demonstrates that this GPA1va5s0-produced phenotype requires the mating-factor receptor and the 13 subunit of the G protein. The double mutant of sst2-1 GPAI Va-50 recovered from division arrest, which suggested that SST2 is not required for recovery of the GPAJVal-50 mutant.G proteins are a family of guanine nucleotide-binding proteins known to be involved in various transmembrane signaling systems in eucaryotic cells (7,27). They consist of three subunits, at, 1B, and y, in mammalian systems. The a subunit contains a guanine nucleotide-binding site and determines the specificity of the protein for its receptor and effector. The a and y subunits form a tightly associated f3y complex. The function of G proteins is regulated by utilizing a guanine nucleotide-binding and hydrolysis cycle. The binding of ligand to its receptor induces GDP-GTP exchange on the a subunit, resulting in dissociation of the GTP-bound a subunit from the ,By complex. The released a subunit, with GTP bound, is then able to interact with a specific effector and regulate its function. After hydrolysis of GTP to GDP by an intrinsic GTPase, the ax subunit reassociates with the ,By complex, regenerating the inactive G protein (7,27).

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