Cloning, characterization, and mapping of the gene encoding the human G protein gamma 2 subunit

G proteins play vital roles in cellular responses to external signals. The specificity of G protein-receptor interaction is mediated mostly by the gamma-subunit and the individual members of the gamma-subunit multigene family would hence be expected to each have a particular expression profile. In an experiment designed to isolate genes expressed predominantly in human testis we identified a cDNA fragment corresponding to the gamma2 gene. Although the protein sequence of the gamma2 subunit has previously been published, the cDNA sequence, expression pattern, genomic structure, and localisation of the human GNG2 gene have not been described. We report the complete sequence of the GNG2 cDNA which is 1066 bp long and contains an open reading frame encoding a protein of 71 amino acids. This protein is 100% homologous to the bovine, mouse, and rat G protein gamma2 subunit. The gene structure is very similar to that of other Ggamma-subunit genes in that there are two introns, one located in the 5' UTR and the other within the ORF. We show that this gene is expressed in a range of foetal tissues as well as adult testis, adrenal gland, brain, white blood cells and lung but not in adult liver, muscle, sperm, prostate gland nor in the testes of two different infertile patients. There is evidence that GNG2 is expressed in malignant tissues. Using two independent methods, we have mapped the human GNG2 gene to chromosome 14q21.     

Gamma-subunit of mouse retinal cyclic-GMP phosphodiesterase: cDNA and corresponding amino acid sequence

The cDNA nucleotide and corresponding amino acid sequences of the gamma-subunit of cyclic-GMP phosphodiesterase (cGMP-PDE gamma) from mouse retina have been determined. The cDNA translated region was found to be 91.5% homologous to the cDNA coding region for the enzyme from bovine retina [(1986) FEBS Lett. 204, 288-292]. On Northern blots of normal mouse retinal RNAs this cDNA hybridized the cGMP-PDE gamma mRNA which is 900 bp long. The mouse gamma-subunit contains 87 amino acid residues which share 97.7% homology with the bovine polypeptide [(1986) FEBS Lett. 204, 288-292]. Only two amino acids have been changed, Ala 8 to Gly and Met 17 to Ile.     

Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning.

The GTP-binding regulatory proteins (G proteins) that transduce signals from receptors to effectors are composed of alpha, beta, and gamma subunits. Whereas the role of alpha subunits in directly regulating effector activity is widely accepted, it has recently been demonstrated that beta gamma subunits may also directly regulate effector activity. This has made clear the importance of identifying and characterizing beta and gamma subunits. We have isolated a cDNA clone encoding a new gamma subunit, referred to here as the gamma 7 subunit, using probes based on peptide sequences of a gamma subunit previously purified from bovine brain. The clone contains a 1.47-kilobase cDNA insert, which includes an open reading frame of 204 base pairs that predicts a 68-amino acid polypeptide with a calculated M(r) of 7553. The predicted protein shares amino acid identities with the other known gamma subunits, ranging from 38 to 68%. Also characteristic of gamma subunits is a carboxyl-terminal CAAX motif. The expression of the gamma 7 subunit as well as the gamma 2, gamma 3, and gamma 5 subunits was examined in several bovine tissues at both the mRNA and protein levels. Whereas the gamma 2 and gamma 3 subunits were selectively expressed in brain, the gamma 5 and gamma 7 subunits were expressed in a variety of tissues. Thus, the gamma 5 and gamma 7 subunits are the first G protein gamma subunits known that could participate in the regulation of widely distributed signal transduction pathways.     

Existence of two gamma subunits of the G proteins in brain

Although amino acid sequences have been determined for the alpha and beta subunits of Gs, Gi, and Go, sequences have not been reported for the gamma subunits of these G proteins. In the present paper, we determined the sequences of peptides prepared by partial proteolysis of two different forms of the gamma subunit of Gs, Gi, and Go from bovine brain. Using oligonucleotide probes based on the sequences of two of these peptides, a cDNA clone was isolated from a bovine adrenal cDNA library. This clone contained a 0.9-kilobase cDNA insert that included an open reading frame of 213 bases encoding a 71-amino acid polypeptide with an estimated Mr of 7850. The amino acid sequence predicted for the adrenal cDNA clone was identical to that determined for one form of the gamma subunit from brain. In addition, an antibody to a peptide based on the predicted amino acid sequence of this cDNA clone reacted specifically with one of the brain gamma subunits, indicating the adrenal cDNA clone encodes a gamma subunit present in both adrenal gland and brain. Also, evidence is presented, demonstrating the existence of a second, structurally distinct, form of the gamma subunit of Gs, Gi, and Go in brain.     

Molecular cloning of a new human G protein. Evidence for two Gi alpha-like protein families

The amino acid sequence of a novel G protein alpha subunit (Gx alpha) has been deduced from the nucleotide sequence of a human cDNA clone isolated from a differentiated HL-60 cDNA library. The cDNA encodes a polypeptide of 354 amino acids (Mr 40,519) which is closely related to Gi alpha proteins. The amino acid sequence homology between Gx alpha and human myeloid Gi alpha is 86% with 15 nonconservative substitutions. Gx alpha also shares 86% homology with both rat brain and mouse macrophage Gi alpha but is more homologous (94%) to bovine brain Gi alpha with only 5 nonconservative amino acid differences. G proteins previously termed Gi alpha may fall into at least two distinct groups, with one including human myeloid Gi alpha, rat brain Gi alpha and mouse macrophage Gi alpha; and other Gx alpha and bovine brain Gi alpha. One group probably contains true Gi and the other a new class of G protein whose function remains to be determined.     

G-protein beta gamma forms: identity of beta and diversity of gamma subunits

Signal-transducing G-proteins are heterotrimers composed of GTP-binding alpha subunits in association with a tightly bound complex of beta and gamma subunits. While the alpha subunits are recognized as a family of diverse structures, beta and gamma subunits have also been found as heterogeneous isoforms. To investigate the diversity and tissue specificity of the beta gamma complexes, we have examined homogeneous oligomeric G-proteins from a variety of sources. The beta and gamma subunits isolated from the major-abundance G-proteins from bovine brain, bovine retina, rabbit liver, human placenta, and human platelets were purified and subjected to biochemical and immunological analysis. Protease mapping and immune recognition revealed an identical profile for each of the two distinctly migrating beta isoforms (beta 36 and beta 35) regardless of tissue or G-protein origin. Digestion with V8 protease revealed four distinct, clearly resolved terminal fragments for beta 36 and two for beta 35. Trypsin and chymotrypsin digestion yielded numerous bands, but again each form had a unique profile with no tissue specificity. Tryptic digestion was found to be conformationally specific with the most resistant structure being the native beta gamma complex. With increasing trypsin, the complex was digested but in a pattern distinct from that for denatured beta. In contrast to the two highly homologous beta structures, examination of this set of proteins revealed at least six distinct gamma peptides. Two unique gamma peptides were found in bovine retinal Gt and three gamma peptides in samples of bovine brain derived Go/Gi. Human placental and platelet Gi samples each contained a unique gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a probe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev-1 proteins and smg p21B is identical with the rap1B protein.     

Amino acid sequence of the human protein synthesis initiation factor eIF-4 gamma.

Eukaryotic protein synthesis initiation factor (eIF) 4 gamma, also known as p220, is a component of the protein complex eIF-4, which is involved in the recognition of the mRNA cap, ATP-dependent unwinding of 5'-terminal secondary structure and recruitment of mRNA to the ribosome. Peptide sequence data from rabbit reticulocyte eIF-4 gamma was used to synthesize oligonucleotide probes and polymerase chain reaction primers. These were used to screen lambda-cDNA libraries from rabbit and human brain, yielding a partial rabbit and a complete human cDNA sequence of 5.1 kilobases. Northern blot and primer extension analysis indicated that the cDNA sequence was complete. To confirm that the cDNA represented that of eIF-4 gamma, three peptides were synthesized based on cDNA sequences and used to produce anti-peptide antibodies. The antibodies specifically recognized intact eIF-4 gamma and its cleavage products following poliovirus infection. The eIF-4 gamma mRNA contains AUG codons at nucleotides 6, 67, 90, 165, and 369, but only the last is followed by a long open reading frame. The eIF-4 gamma polypeptide is 154 kDa (1396 amino acid residues) and contains sequence motifs of potential interest: a sequence (AGLGPR) that is similar to the substrate recognition sequence of protease 2A from rhinovirus serotype 14, five PEST regions with scores greater than 10, which are characteristic of rapidly degraded proteins, stretches of polyglutamic acid, and numerous potential phosphorylation sites.     

Somatic cell mapping and restriction fragment analysis of bovine genes for fibronectin and gamma crystallin

DNAs from cow-hamster and cow-mouse somatic hybrid cells segregating bovine chromosomes have been analyzed by Southern blotting and hybridization with human fibronectin and gamma crystallin probes. Concordancy of retention of these bovine genes was compared to cattle isozyme loci representing previously described syntenic groups. Bovine fibronectin (FNI) and gamma crystallin (CRYG) fragments were concordant with each other and with isocitrate dehydrogenase 1 (IDH1), representing the bovine syntenic group U17. The syntenic relationship of these genes is conserved on human chromosome 2q and also on mouse chromosome 1. In addition, bovine RFLPs were identified with both fibronectin and gamma crystallin probes. These polymorphisms will be used to study recombination between the syntenic loci in pedigreed herds and to mark a segment of the bovine genome that is likely homologous to the Lsh region of mouse chromosome 1, which confers resistance in mice to several intracellular parasites.     

The gamma-subunit of ATP synthase from spinach chloroplasts. Primary structure deduced from the cloned cDNA sequence

cDNA clones encoding the gamma-subunit of chloroplast ATP synthase were isolated from a spinach library using synthetic oligonucleotide probes. The predicted amino acid sequence indicated that the mature chloroplast gamma-subunit consists of 323 amino acid residues and is highly homologous (55% identical residues) with the sequence of the cyanobacterial subunit. The positions of the four cysteine residues were identified. The carboxyl-terminal region of the chloroplast gamma-subunit is highly homologous with those of the gamma-subunits from six other sources (bacteria and mitochondria) sequenced thus far.     

Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits.

We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743-748]. To investigate whether this stimulation was due to a soluble alpha subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein beta gamma dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that beta gamma subunits, purified from bovine retinal transducin (beta gamma t), markedly stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by beta gamma t was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin alpha subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. Beta gamma t also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to beta gamma t, but was also observed with highly purified beta gamma subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL-60 granulocytes. Only one of these forms was sensitive to stimulation by beta gamma t, demonstrating that stimulation of phospholipase C by beta gamma subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein beta gamma subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.     

Molecular cloning of rabbit gamma heavy chain mRNA.

A cDNA library of rabbit spleen mRNA was screened for immunoglobulin heavy chain sequences. In this paper we report the nucleotide sequence of two cDNA clones containing part of the constant region of the rabbit gamma heavy chain mRNA. The sequence encodes part of the CH2 domain (amino acids 268 to 340), the entire CH3 domain (amino acids 341 to 447) and the 3' untranslated region. This nucleotide sequence has been compared to the corresponding sequences of mouse gamma 1, gamma 2a and gamma 2b genes. The homologies between rabbit gamma chain gene sequence and each of the mouse gamma chain gene sequences are of the same magnitude order. This comparison shows that the CH2 domains are more homologous to each other than CH3 domains or 3' untranslated sequences. The presence of species specific nucleotide positions suggests that mouse gamma chain genes could have evolved from a common ancestor shortly after the mouse-rabbit species separation. Genomic blot analysis of rabbit liver DNA with the rabbit C gamma probes shows a limited number of related sequences, with little restriction site polymorphism between individual rabbits.     

The alpha- and beta-subunits of the human UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase [corrected] are encoded by a single cDNA

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed. Although cDNA encoding the gamma-subunit has been described, cDNAs for the alpha- and beta-subunits have not. Using partial amino acid sequences from the bovine alpha- and beta-subunits, we have isolated a human cDNA that encodes both the alpha- and beta-subunits. Both subunits contain a single predicted membrane-spanning domain. The alpha- and beta-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the alpha/beta-subunits-precursor cDNA with or without the gamma-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the alpha-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.     

Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.     

Cloning and sequencing of a cDNA for human mitochondrial ubiquinone-binding protein of complex III

The ubiquinone-binding protein (QP-C) is a nuclear-encoded component of ubiquinol-cytochrome c oxidoreductase in the mitochondrial respiratory chain and plays an important role in electron transfer as a ubiquinone-QP-C complex. We obtained a partial cDNA for rat liver QP-C by screening a lambda gt11 rat liver cDNA library using antiserum directed against bovine heart QP-C. Using this cDNA as a probe, a cDNA clone was isolated from a human fibroblast cDNA library by colony hybridization. The total length of the cloned cDNA was 518 base pairs with an open reading frame of 333 base pairs. The 111-amino acid sequence deduced from the nucleotide sequence of the cDNA is 85% homologous to that of bovine QP-C and contains only a single additional amino-terminal methionine. This implies that the human QP-C is synthesized without a presequence which is required for import of most nuclear-encoded mitochondrial proteins into mitochondria.     

Cloning, expression analysis and chromosome mapping of human casein kinase 1 gamma1 (CSNK1G1): identification of two types of cDNA encoding the kinase protein associated with heterologous carboxy-terminal sequences

Casein kinase 1 gamma1(CK1 gamma1) is known to be involved in the growth and morphogenesis of eukaryotic cells. We have isolated two types of cDNA for human casein kinase 1 gamma1 (hCK1 gamma1). One of them (hCK1 gamma1S) was found to encode a polypeptide consisting of 393 amino acids, which is highly homologous with already reported rat CK1 gamma1 (rCK1 gamma1). The other type of cDNA (hCK1 gamma1L) encodes a polypeptide consisting of 422 amino acids, which is quite identical in the kinase domain, but different in the C-terminal sequence from hCK1 gamma1S. Namely, hCK1 gamma1L has a characteristic sequence of 50 amino acids at the C-terminal end and this motif was shown to be shared by the casein kinase gamma2 and gamma3 from rat and human, suggesting that it is a signature sequence of the gamma-isoforms. In this sense, newly isolated hCK1 gamma1L might be the original form of CK1 gamma1 subspecies rather than rCK1 gamma1 and hCK1 gamma1S. RT-PCR analysis revealed that hCK1 gamma1S mRNA is predominantly present in the testis, whereas the abundance of hCK1 gamma1L mRNA was nearly the same in the twelve tissues examined. These results suggest that novel hCK1 gamma1L may have a unique functional role different from that of hCK1 gamma1S and rCK1 gamma1. The human hCK1 gamma1 gene (CSNK1G1) was mapped to chromosome 15q22.1-->q22.31 by fluorescence in situ hybridization.     

cDNA clones encoding bovine gamma-crystallins

We have determined the nucleotide sequence of two bovine lens gamma-crystallin cDNA clones, pBL gamma II-1 and pBL gamma III-1. The 644 bp cDNA insert of pBL gamma II-1 contains coding information for the entire amino acid sequence of bovine gamma II-crystallin. The 497 bp cDNA insert of pBL gamma III-1 encodes a homologous but different gamma-crystallin polypeptide, and appears to lack the coding information for the C-terminal 17 amino acid residues. While the nucleotide and predicted amino acid sequences of the coding regions of the clones show a high degree of homology, the untranslated leader sequences are relatively dissimilar. The leader sequence of pBL gamma III-1 is strikingly homologous to a portion of a rabbit immunoglobulin alpha-heavy chain mRNA.     

The structure and expression of the guinea pig Fc receptor for IgG1 and IgG2 (Fc gamma 1/gamma 2R).

A cDNA clone encoding the receptor for guinea pig immunoglobulin G was isolated from a guinea pig peritoneal macrophage cDNA library. The cloned cDNA encoded 271 amino acids containing an N-terminal signal sequence. The deduced amino acid sequence is most homologous to murine Fc gamma RII beta 2. The receptor protein could be expressed in COS-7 and L cells transfected with the cDNA, suggesting that the expression of this receptor does not require the co-expression of a second chain such as gamma chain of Fc epsilon RI or CD3 zeta chain. The transformant L cells showed the binding to both the guinea pig IgG1 and IgG2 antibodies complexed with antigen, indicating that the cDNA we cloned was the one for guinea pig Fc gamma 1/gamma 2R.     

Amino acid sequence of bovine gamma E (IVa) lens crystallin.

When electrospray ionization mass spectrometry (ESMS) was used to analyze purified bovine gamma E (gamma IVa)-crystallin, it yielded a relative molecular mass (M(r)) of 20.955 +/- 5. This mass is significantly different from that calculated from the published sequence (M(r) 20.894) (White HE et al., 1989, J Mol Biol 207:217-235). Further, ES-MS analysis of the protein after it had been reduced and carboxymethylated indicated the presence of five cysteine residues, whereas the published sequence contains six (Kilby GW et al., 1995, Eur Mass Spectrom 1:203-208). The entire protein sequence of gamma E crystallin has therefore been studied via a combination of ES-MS, ES-MS/MS, and Edman amino acid sequencing. The corrected sequence gives an M(r) of 20.955.3, which matches that obtained by ES-MS analysis of the purified native protein. The corrected sequence is also in agreement with a recent cDNA sequence obtained for a bovine gamma-crystallin by R. Hay (pers. comm.).