Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain.

Abstract

We have cloned cDNAs encoding a subunits of the guanine nucleotide-binding proteins G., G,, and G. and determined their nucleotide sequences. Purified preparations of G, and G. a subunits (Gia and G~a) from rat brain were completely digested with trypsin, and peptides were subjected to amino acid sequence analysis. By screening of a cDNA library from rat C6 glioma cells with a synthetic probe corresponding to a 17 amino acid sequence, a clone encoding the sequence of G~a was obtained. Then, the library was rescreened with a G~a cDNA probe to isolate several strongly or weakly hybridizing clones. cDNAs encoding the complete sequences of Gia and Gsa were thus obtained. From nucleotide sequence analysis, the amino acid sequences of Gsa and Gla were deduced; they contain 394 and 355 amino acid residues (including the initiator methionine), respectively. The calculated molecular weights for Gsa and Gla were 45,663 and 40,499, respectively. The Goa clone encoded a sequence of 310 amino acid residues that lacked the NH2 terminus. The

Characterization of four GO‐type proteins purified from bovine brain membranes

“…2, all of the four a-subunits were similarly reacted with anti-c~o antibody (B) which was raised against synthetic oligopeptide (EYGDKERKADSK-MVC) corresponding to the predicted amino acid sequence (94-108) of ozo [3]. The sequence in C~o was the most heterogeneous region among G proteins [3], and the antibody did not crossreact with the other types of o~-subunits, such as c~i, c~s, or c~t (data not shown).…”

“…Immunoblot analysis was performed as in [ 11,15,16]. Antibodies used in the present experiments were anti-OLo (A) which was affinity-purified rabbit polyclonal IgG raised against purified O~o and anti-c~o (B) which was also affinity-purified rabbit polyclonal antibody raised against synthetic oligopeptide corresponding to the predicted amino acid sequence (94-108; EYGDKERKADSKMVC) of OLo [3]. This region is specific for C~o and different from the other oL-subunits of G proteins.…”

“…The cr subunits of Gi serve as the substrate of pertussis toxin-(lAP-) catalyzed ADP-ribosylation in their trimeric forms, have been classified into three subtypes of Oti-1, O/i-2 and o'i-3 from analyses of molecular cloning of the genes [2] and cDNAs [3][4][5][6]. In regard to the cesubunits of Go, at least two types of the genes were informed from Northern blot analysis [7].…”

Isolation and characterization of the human Gs alpha gene.

“…About 3 x i0s plaques of human genomic library (14) were screened with 32P-labeled rat Gsa as a probe (4 (5) with the sequence of the human Gsa gene suggests that four types of Gsa mRNAs may be generated from a single Gsa gene by alternative splicing as shown in Fig. 4.…”

“…The a subunits of G protein (Ga) bind guanine nucleotide and are unique to each G protein. Recent progress in molecular cloning of Ga cDNAs revealed that they are highly similar proteins (4). So far, four Gsa (5), three Gia (4,6,41), two Gta (7), and Goa (4) cDNAs were isolated and their sequences were determined (see also ref.…”