XLalphas, the extra-long form of the alpha-subunit of the Gs G protein, is significantly longer than suspected, and so is its companion Alex

Abstract

Because of the use of alternate exons 1, mammals express two distinct forms of Gsalpha-subunits: the canonical 394-aa Gsalpha present in all tissues and a 700+-aa extra-long alphas (XLalphas) expressed in a more restricted manner. Both subunits transduce receptor signals into stimulation of adenylyl cyclase. The XL exon encodes the XL domain of XLalphas and, in a parallel ORF, a protein called Alex. Alex interacts with the XL domain of XLalphas and inhibits its adenylyl cyclase-stimulating function. In mice, rats, and humans, the XL exon is thought to contribute 422.3, 367.3, and 551.3 codons and to encode Alex proteins of 390, 357, and 561 aa, respectively. We report here that the XL exon is longer than presumed and contributes in mice, rats, and humans, respectively, an additional 364, 430, and 139 codons to XLalphas. We called the N-terminally extended XLalphas extra-extra-long Gsalpha, or XXLalphas. Alex is likewise longer. Its ORF also remains open in the 5' direction for approximately 2,000 nt, giving rise to Alex-extended, or AlexX. RT-PCR of murine total brain RNA shows that the entire XXL domain is encoded in a single exon. Furthermore, we discovered two truncated forms of XXLalphas, XXLb1 and XXLb2, in which, because of alternative splicing, the Gsalpha domain is replaced by different sequences. XXLb proteins are likely to be found as stable dimers with AlexX. The N-terminally longer proteins may play regulatory roles.

Fig. 1.

Intronexon organization of the murine Gnas complex locus at the distal end of chromosome 2, orientation of the transcription units, and allelic expression characteristics as known before the present studies. (A) Intronexon organization. Neither exons (boxes) nor introns (interbox distances) are to scale; however, their relative positions reflect their positions in the genome. Exact parameters can be obtained from GenBank genomic contig NT_039211, region 3831406-3893830. p (paternal), m (maternal), and b (biallelic) next to arrows depicting promoter location and direction indicate a transcribed allele, where p transcripts are maternally silenced (imprinted) and m transcripts are paternally imprinted. (B) Main fully processed transcripts and their translation products. Two, 1A and Nespas, appear to be noncoding. *, Stop codon.

Fig. 2.

The genomic sequence of mouse distal chromosome 2 encodes extended XL and Alex domains, XXL and AlexX, that are potentially much longer than suspected. Analysis of the mouse genomic DNA sequence (GenBank accession no. NT_039211, region 3844806-3847406), which encodes the XL and Alex domains of the Gnas1 complex locus, shows the existence of ORFs of 785.3 and 725 codons that run in parallel and encode in their 3′ end the murine XL and Alex domains of the 422.3-codon XL and 391-codon Alex domains reported in GenBank's AF116268 record. Shown are the nucleotide sequence and, above, the amino acids encoded in the two ORFs. Nucleotides in uppercase are from AF116268 (cDNA), as well as from NT_039211 (genomic sequence); nucleotides in lowercase are from NT_039211 only; nucleotides in italics are from intronic sequences. Amino acids in uppercase indicate the XL domain and its potential N-terminal extension, referred to as XXL; amino acids in lowercase indicate Alex and its potential N-terminal extension, referred to as AlexX (Fig. 4). Also shown, in bold and uppercase characters, are the forward (F) and reverse (R) PCR primers XXF, XF, X3F, and X5R (Fig. 4), which were used to test for the existence of the XXL/AlexX domains.

Fig. 3.

Amino acid sequence alignments of the extended mouse, rat, and human XXL (A) and AlexX (B) domains as deduced on the basis of RT-PCR analysis of mouse brain transcripts and mouse, rat, and human genomic DNA sequences accessible at www.ncbi.nih.gov. Amino acid sequences are based on: (i) the RT-PCR analysis reported in this study (Fig. 4); (ii) GenBank cDNA records X84047 (rat XLαs), AF116268 (mouse XLαs), AK030489 (mouse XLb), and NM_080425 (human XLαs); and (iii) orthologous sequences found in genomic contigs NT_039211 (mouse), NT_011362 (human), and NW_047679 (rat). (A) Alignment of XXL amino acid sequences; (B) Amino acid alignment comparing variations in sequence in the region coded for by exons lying between the end of the XXL exon and Gnas1 exon 2; (C) Alignment of AlexX amino acid sequences. The key to alignments follows. First line: master sequence to which the other sequences are compared. Second and third lines: -, identical to the master sequence; uppercase letter, different from the master sequence. Consensus sequence (bold): uppercase letters, amino acids identical in all sequences compared; -, different by at least one from the master sequence. *, Stop codon.

Fig. 4.

RT-PCR analysis of total mouse brain RNA shows presence of transcripts derived from the 5′ end of the extended XXL/AlexX ORFs depicted in Fig. 3. (A) Diagrams of proven and potential XL-related transcripts and approximate location of primers used to test for their existence. Primers XXF, XF, X3F, and X5R are defined in Fig. 3. Other primers were as follows: XLb1R, <-3′-CCAAT GGAAC ACTAA ACGCG GTAC-5′; Ex2R, <-3′-CTCAG ACCGT TTTCG TGGTA TGGTA ACAC-5′; Ex12R, <-3′-ACCTG TGACT CTTGT AGGCG GCACA-5′. Blue diagonal numbers indicate codons of ORFs. (B) Agarose gel electrophoresis of PCR products: XXL and AlexX exist, and XXL splices to Gnas1 exon 2 either directly, giving XXLαs, or via an intermediary alternative exon, XLb, giving rise to XXLb2. Note that the PCR analysis shows the presence of a splice variant that gives rise to XXLb2, whose mRNA differs from that of XXLαs in that it incorporates 95 nt of the XXLb exon. Lanes: 1-5, PCR products obtained by using reverse-transcribed single-stranded cDNAs as templates; 6-12, PCR products obtained by using the reaction products of a mock single-stranded cDNA synthesis reaction containing the RNA template but obtained by omitting the reverse transcriptase. Absence of products indicates absence of contaminating DNA that could have been used as template in the reactions shown in lanes 2-6.

Fig. 5.

Nucleotide sequence of the region coding for Gsα amino acids Met-221 through Arg-280, which in Homo sapiens are encoded in exons 9 and 10, reveals them to be encoded in a single exon in Mus musculus. (A) The genomic region of interest was amplified by PCR (forward and reverse primers as shown) using genomic DNA from an FVB/N mouse and sequenced by the dideoxynucleotide chain termination method. Shown is the complete sequence of the amplified DNA and the deduced amino acid sequence encoded in the fully spliced RNA. This sequence (only partially shown) spans from the middle of exon 7 to the middle of the intron that follows the exon 9-10 coding region. The sequence shown for the murine gene was identical for DNA from 129SV, FVB/N, BALB/c, and C57BL/6J mice. (B) Human Gnas1 nucleotide and deduced amino acid sequence (ref. ; GenBank accession no. NT_011362 region). Nucleotide sequences (in blocks of 10): uppercase, coding (exonic); lowercase italic, noncoding (intronic).

Fig. 6.

Update of splice patterns (A) and translation products (B) under control of the XXL promoter.