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Biology of Reproduction 60, 1528-1535 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Identification and Expression of Go1 and Go2 {alpha}-Subunit Transcripts in Human Myometrium in Relation to Pregnancy

P. Duc-Goiran1,a, C. Bourgeoisa, T.M. Mignota, B. Roberta, G. Tanguya, and F. Ferréa

a INSERM U. 361, Université René Descartes Paris V, Pavillon Baudelocque, 75014 Paris, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The 39-kDa Go{alpha} protein, the {alpha} subunit of a major heterotrimeric G protein of brain and neuroendocrine cells, was found to be present in human myometrium. Using three different antisera, we showed its strong expression in myometrium from pregnant patients as compared to nonpregnant ones. This is in agreement with the high expression level of its two isoforms ({alpha}o1 and {alpha}o2), previously described in late pregnancy. To better ascertain the nature of these immunoreactive isoforms, we investigated transcripts of the Go{alpha} gene in myometrium from pregnant and nonpregnant patients by reverse transcription-polymerase chain reaction (RT-PCR). In this tissue, the amplified cDNA product of a region common to both Go1{alpha} and Go2{alpha} mRNA variants was recognized as the Go{alpha} nucleotide sequence. Transcripts of Go1{alpha} and Go2{alpha} were identified by sequencing. A partial cDNA Go2{alpha} sequence was described, which differed from the Go{alpha} gene by two nucleotides in exon 8B. Levels of Go1{alpha} and Go2{alpha} transcripts analyzed by semi-quantitative RT-PCR were significantly higher in myometrium from pregnant than from nonpregnant patients. It is suggested that Go{alpha} gene expression in this tissue may contribute to modifications seen in the signaling pathways observed at the end of pregnancy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During pregnancy, development and contractile activity of uterine muscle are under the control of numerous extracellular signals, in particular, hormones, neurotransmitters, autocoids. Most of these factors act on their myometrial cellular targets via 7-transmembrane segment receptors coupled to heterotrimeric G-proteins that transfer the signal to a variety of effector systems including enzymes and ion channels, leading to second-messenger generation [1, 2]. The G protein heterotrimer consists of an {alpha} subunit that binds and hydrolyses GTP and a pair of tightly associated ß and {gamma} subunits. Both the free {alpha} and the ß{gamma} dimer independently regulate intracellular effectors [3]. Each subunit forms a family of varying degrees of complexity, and highly defined combinations of {alpha}ß{gamma} subunits seem to be required for G protein function [4]. The {alpha} subunits comprise a large family of homologous proteins containing discontinuous regions, designated G-1 to G-5, which are highly conserved across the GTPase superfamily. These regions are critical in GDP/GTP exchange, GTP-induced conformational change, and GTP hydrolysis, mechanisms widely shared among members of this superfamily [5].

G proteins are classified according to their {alpha} subunits and fall into four major classes (G{alpha}s, G{alpha}i, G{alpha}q, G{alpha}12) based on similarities of amino acid sequences. Many, but not all, G{alpha} subunits are the targets of two bacterial ADP-ribosylating exotoxins: cholera toxin (CTX) and pertussis toxin (PTX). GS, Golf, and transducin (Gt) are CTX substrates, whereas a wide spectrum of G proteins that constitute the Gi/Go family are ADP-ribosylated by PTX. However, transducins and the Gz protein, which are resistant to this toxin, are included in this family, because of the homology of their DNA sequences.

Among the PTX-sensitive G{alpha} proteins detected so far in human myometrium during pregnancy [6] is a 39-kDa protein that resembles the Go{alpha} protein found almost exclusively in nervous tissue and neuroendocrine cells [7]. In rodent [8–10] and bovine species [11], two isoforms of the {alpha}o subunit ({alpha}o1 and {alpha}o2), which differ by 26 amino acids dispersed along the last 111 amino acids of their C-terminal region, can be generated by alternative splicing of exons 7 and 8 of the Go{alpha} primary transcript. Their expression varies widely with differentiation [12], organ development [13] and hormone treatment [14]. In humans, the sequence of the unique Go{alpha} gene is known [10, 15], but only partial Go1{alpha} cDNAs from brain have been characterized. The Go1{alpha} cDNA described by Lavu et al. [16] is truncated at the 5' and 3' ends. As yet, only the spliced 3' untranslated regions (UTR-A and -B) of Go1{alpha} cDNAs have been characterized, by Murtagh et al. [15].

In late pregnancy, an increase in the expression of Go1{alpha} and Go2{alpha} protein isoforms has been shown in human myometrium using antibodies against dodecapeptides from the more divergent region of the {alpha}o1 and {alpha}o2 deduced amino acid sequences of the rodent Go{alpha} gene [17]. In order to assess the nature of these immunoreactive isoforms, we compared their expression with that of transcripts in human myometrium. First, we analyzed transcripts of a Go{alpha} region common to both isoforms by specific reverse transcription (RT)-polymerase chain reaction (PCR), by nested PCR, and by direct sequencing. Secondly, we assessed the presence of Go1{alpha} and Go2{alpha} transcripts and described their sequences. Finally, we analyzed the expression of both transcripts in relation to pregnancy by semiquantitative RT-PCR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Electrophoretic reagents were provided by Bio-Rad (Richmond, CA). All substances used for RNA isolation were molecular reagents from Sigma Chemical Co. (St. Louis, MO). 125I-Protein A (30 mCi/mg), [{gamma}32-P]ATP (3000 Ci/mmol), Hybond-C and Hybond-N+ membranes, the enhanced chemiluminescence (ECL) 3'-oligolabeling and detection system, and x-ray films were obtained from Amersham Pharmacia Biotech (Saclay, France).

Tissues and Cells

Human myometrium Myometrial biopsies were obtained from patients with normal uncomplicated pregnancies who delivered by elective cesarean section, indicated for diagnosed cephalopelvic disproportion or previous cesarian sections. Sections were done between the 37th and 38th week of pregnancy when the women were not in labor. Samples of myometrium were taken from the upper border of the uterine incision. Informed consent was obtained beforehand. This study was approved by the Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale (CCPPRB) de Paris-Cochin. Healthy myometrial samples were also taken from cycling patients undergoing total hysterectomy for benign gynecological disorders such as metrorrhagia or fibromyomas. All myometrial samples were taken in the longitudinal layer. They were collected on ice, dissected free of serosa, and quickly frozen at -80°C until processed.

Human cervical myofibroblasts Human myofibroblasts were obtained from uterine cervical biopsies in nonpregnant patients and cultured by the explant method [18].

Rat brain Brains were obtained after decapitation from Wistar female rats aged 80 days and stored at -80°C until membrane fraction preparation.

Preparation of Membrane Fractions and Immunoblotting

Tissue samples were processed as previously described [19]. Briefly, membrane fractions alkylated with N-ethyl-maleimide were resolved by SDS-PAGE (10% w:v), electrotransferred to a nitrocellulose membrane, and probed with antisera. All antisera were polyclonal antibodies raised in rabbits. Antisera GC/2 and Go/1 were from New England Nuclear-Dupont (Boston, MA). GC/2 antiserum was raised against the N-terminal peptide (2–17) from rat Go{alpha} protein, while Go/1 antiserum was raised against a decapeptide corresponding to the C-terminal amino acids (345–354) of human Go{alpha} protein [20]. Antibodies against Go{alpha} protein purified from bovine brain were a kind gift from Dr. B. Rouot (INSERM, U.431). Antigen-antibody complexes were revealed by 125I-protein A and detected by autoradiography.

RNA Extraction and RT

Total RNA was extracted from myometrial biopsies or cell cultures using an acidic guanidinium thiocyanate-phenol-chloroform procedure [21]. RT was performed as previously described [22], with 2 µg of total RNA, using random hexanucleotides (20 µM) as primers and 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Cergy Pontoise, France). A control reaction was conducted without reverse transcriptase in each experiment. Total RNAs from human normal adult pituitary gland and the human DAMI megakaryoblastic cell line were used as positive and negative controls, respectively. They were gifts from Y. De Keyser (pituitary; Institut Cochin de Génétique Moléculaire, Paris) and M. Mitjavila (DAMI; INSERM, U. 362, Villejuif, France).

DNA Amplification

Sequences of primers and probes are shown in Table 1. External sense and antisense oligonucleotides were selected in separate exons of the known human Go{alpha} gene [10]. Within these exon regions, we determined the Go{alpha} oligonucleotide sequence that was least homologous with Gs, Gi, Gz, and G12 {alpha} subunits. Human ß2-microglobulin (Clontech, Ozyme, France) [23] and human 28S ribosomic RNA [24, 25] amplimer sets were used to verify the integrity of cDNA preparations.


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  		TABLE 1. Oligonucleotide sequence of primers and probes.

PCR was performed as in [22]. Amplification was performed at a varying number of cycles of sequential steps: 1 min at 94°C, 1 min at 52.8°C for PCR-1 (52.3°C for PCR-2, 54.9°C for PCR-3, 59.5°C for PCR-4, 60.5°C for ß2-microglobulin), and 1 min at 72°C. Sequential steps for 28S ribosomic RNA amplification were as in [24]. RT-PCR products were separated on a 3% NuSieve GTG agarose gel and visualized by ethidium bromide staining. The specificity of each amplification product was checked by Southern blot analysis [22]. The hybridization was performed with a specific internal oligonucleotide labeled with [{gamma}32]ATP or fluorescein-11-deoxy-UTP using an ECL 3'-oligolabeling and detection system kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

For semiquantification of Go{alpha} transcripts, we first defined the correct number of cycles to use before quantification and determined the optical density equivalent to half of the maximal response (OD50). The linearity of signals obtained in amplification of each DNA fragment can be demonstrated in a range of cycles depending on the transcript levels of each gene. Ten myometrial RNA samples, 5 from nonpregnant patients and 5 from pregnant patients, were subjected to RT in the same experiment. They were amplified for 25 cycles with Go{alpha} primers of the common region of Go{alpha} gene, for 26 cycles with Go1{alpha} and Go2{alpha} primers, for 20 cycles with ß2-microglobulin primers, and for 10 cycles with 28S ribosomic primers.

Polaroid pictures of the ethidium bromide staining gel containing the ten myometrial RNA samples were scanned with a StudioScan IIsi instrument (Agfa PhotoScan Software, Agfa-Gevaert, Germany). The intensity of the bands were analyzed densitometrically by the Adobe Photoshop 4.0 software package (Mountain View, CA). The relative intensity corresponds to the amount of mRNA calculated by assigning an arbitrary value of 100 to the band of maximal intensity for each gel and establishing a relative index.

Sequencing

After electrophoresis of the PCR product and ethidium bromide staining, the resulting DNA band was excised from the gel, electro-eluted, and ethanol-precipitated. The direct sequencing of the DNA template was performed according to a method based on that of Sanger et al. [26]. The reaction was carried out with a T7 sequencing kit (Amersham Pharmacia Biotech) or in a thermal cycler with a PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer Applied Systems, Foster City, CA), which relies on four dye-labeled dideoxynucleotides as terminators and a thermally stable AmpliTaq DNA polymerase. Extension products were analyzed on the 377A or 373A DNA sequencer (Perkin Elmer Applied Biosystems).

For each sample, sequencing of the PCR fragment obtained was carried out with a sense primer (2 runs minimum) and an antisense primer (2 runs minimum).

Statistical Analysis

The nonparametric Wilcoxon Mann-Whitney test for unpaired samples was applied for comparison of the mRNA expression levels in myometrium of 5 pregnant and 5 nonpregnant women. All results were expressed as the means ± SEM of the percentage of maximum optical density. A p value of less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunodetection of Go{alpha} Protein

Go{alpha} protein from human myometrial membranes was examined using the three polyclonal antibodies mentioned above. The polyclonal GC/2 antiserum raised against a peptide from the N-terminal part of Go{alpha} recognized a 39-kDa protein in myometrium (Fig. 1A, lanes 1 and 2) and in rat brain, the positive control (Fig. 1A, lane 3). Much more intense labeling was observed in pregnant myometrium (Fig. 1A, lane 2) than in myometrium from nonpregnant patients (Fig. 1A, lane 1). Cross-reactivity with a 40-kDa protein was observed in every sample.



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  		FIG. 1. Immunoblot analysis of Go{alpha} protein in myometrium. Membrane fractions (120 µg of proteins) were prepared from myometrium obtained from nonpregnant (lane 1) and pregnant patients near term (lane 2). In lane 3, membrane fractions (10 µg of proteins) were from rat brain. Blot A was probed with GC/2 antiserum, blot B with Go/1 antiserum, and blot C with antibodies against purified bovine Go{alpha} protein.

Using Go/1 antiserum directed against a decapeptide from the C-terminal part of Go{alpha}, which heavily stains the 39-kDa Go{alpha} present in bovine brain extract [27], no immunoreactivity of a 39-kDa protein was observed in nonpregnant myometrial samples (Fig. 1B, lane 1). In contrast, strong immunoreactivity of the 39-kDa protein could be visualized in human myometrium taken from pregnant patients (Fig. 1B, lane 2) as well as in rat brain (Fig. 1B, lane 3). Reactivity with a 40-kDa protein was observed in every myometrial sample at similar levels. No cross-reaction with a 40-kDa protein was observed with rat brain, using a smaller amount of rat membrane (data not shown). Cross-reactivity with a 47-kDa protein was observed in every myometrial sample. However, this protein was not ADP-ribosylated by PTX (data not shown).

Antibodies raised against purified bovine brain Go{alpha} strongly recognized the 39-kDa protein in human myometrium from pregnant patients (Fig. 1C, lane 2) and in rat brain (Fig. 1C, lane 3). This protein was detected very faintly (Fig. 1C, lane 1) in human myometrium from nonpregnant patients. A 40-kDa protein was also labeled by these antibodies in myometrium from both nonpregnant and pregnant patients (Fig. 1C, lanes 1 and 2). No cross-reaction with a 40-kDa protein was observed with rat brain.

Identification of Go{alpha} Transcripts

In order to assess the presence of Go{alpha} protein in myometrium, particularly from nonpregnant patients, we investigated expression of Go{alpha} transcripts. After RT, cDNA was amplified (PCR-1) with external Go{alpha} primers located in a gene region common to both Go1{alpha} and Go2{alpha} (Table 1; Fig. 2C). A 174-base pair (bp) cDNA fragment was amplified from the myometrium of a nonpregnant patient (Fig. 2A, lane 1; Fig. 3A, lane 1). After hybridization with probe 1, an internal antisense oligonucleotide (Table 1; Fig. 2C), a strong signal could be seen (Fig. 2B, lane 6; Fig. 3B, lane 4). No hybridization signal was seen in the absence of reverse transcriptase (Fig. 2A, lane 2; Fig. 2B, lane 7). An amplification product was also obtained in the anterior pituitary gland used as a positive control (Fig. 2A, lane 3). In contrast, no amplification was observed in the megakaryoblastic DAMI cell line (the progenitors of platelets known to be Go{alpha}-negative) (Fig. 2A, lane 4) or in human cervical myofibroblasts (Fig. 2A, lane 5), even after 40 amplification cycles (data not shown). Nevertheless, megakaryoblasts and cervical myofibroblasts expressed Gs{alpha} transcripts, proving the quality of the RT reaction (data not shown). A 174-bp cDNA fragment was also obtained from myometrium of pregnant patients, as can be seen in the more extensive results shown in Figure 6.



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  		FIG. 2. Amplification of a Go{alpha} cDNA region common to both Go1{alpha} and Go2{alpha} transcripts, using external Go{alpha} primers (PCR-1) for 32 cycles. A) RT-PCR products obtained from the myometrium of a nonpregnant patient (lanes 1 and 2), anterior pituitary gland (lane 3), megakaryoblastic DAMI cells (lane 4), and cervical myofibroblasts (lane 5) were visualized by ethidium bromide staining after electrophoresis. Reverse transcriptase was omitted in lane 2. B) Signal obtained after Southern blotting and hybridization with fluorescein-labeled probe 1 in myometrium, in the presence (lane 6) or absence (lane 7) of reverse transcriptase in the RT reaction. C) Diagram of a portion of the Go{alpha} gene. Open boxes represent exons, and interrupted lines represent introns. External sense oligonucleotide is located in exon 3 while the external antisense oligonucleotide is situated in the terminal part of exon 4. Probe 1 is an antisense nucleotide internal to both external primers.



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  		FIG. 3. Specificity of Go{alpha} mRNA expression. The 174-bp cDNA product (A, lane 1 and B, lane 4) amplified from myometrium using Go{alpha} external primers (PCR-1) was used as a template for PCR-2 amplification with nested primers for 32 cycles. A, B) A 130-bp cDNA fragment was obtained (A, lane 2, and B, lane 5). This 130-bp fragment was completely cleaved by BanII (A, lane 3, and B, lane 6). Lanes 1–3: ethidium bromide-stained gel. Lanes 4–6: Southern blot analysis with fluorescein-labeled probe 1. C) Diagram of a portion of Go{alpha} gene. Open boxes: exons; interrupted lines: introns. Nested primers (PCR-2) were internal to PCR-1 primers. The site of BanII hydrolysis (arrowhead) is between nucleotides 356 and 357.



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  		FIG. 6. Distribution of human Go1{alpha} and Go2{alpha} transcripts in myometrium from nonpregnant and pregnant patients. A) RT-PCR products were obtained from myometrial RNA samples, 5 from nonpregnant patients and 5 from pregnant patients. After electrophoresis, they were visualized by ethidium bromide staining and quantified by densitometric scanning. B) After normalization of the values on a 100% scale, relative intensities were shown as means obtained in myometrium from nonpregnant and pregnant patients. *Significant differences between values obtained in nonpregnant and pregnant patients (p < 0.01).

To increase the specificity of the reaction, the 174-bp cDNA fragment amplified from the myometrium was excised from the gel and amplified again (PCR-2) with nested Go{alpha} primers (Table 1; Fig. 3C). A 130-bp cDNA fragment was produced (Fig. 3A, lane 2). It was completely hydrolysed by BanII, a restriction enzyme that cleaves once within this part of the known sequence of Go{alpha} (Fig. 3C), giving rise to two fragments of the predicted size, 65 bp (Fig. 3A, lane 3). After Southern blot analysis and hybridization with probe 1, a strong signal was obtained with the 130-bp fragment (Fig. 3B, lane 5) and a weaker signal with the 65-bp hydrolysis product (Fig. 3B, lane 6). The same results were seen with 130- and 65-bp cDNA fragments from the pituitary gland (data not shown). Finally, the 130-bp cDNA fragment was sequenced and found to be identical to the previously described Go{alpha} cDNA sequence [16].

Identification of Go1{alpha} and Go2{alpha} Transcripts

After RT and amplification with Go1{alpha} primers (Table 1; PCR-3), a 355-bp cDNA fragment was obtained in myometrium from both nonpregnant and pregnant patients (Fig. 4, lanes 5 and 7). No amplification product was observed in the absence of reverse transcriptase (Fig. 4, lanes 6 and 8). After direct sequencing of these 355-bp cDNA fragments, we showed them to have the same nucleotide sequence as Go1{alpha} cDNA previously described in brain [16], with the exception of the third base of codon 339 (GTT). Our data showed this codon to be GTC, as found in genomic Go{alpha} DNA [10] (data not shown). Both GTC and GTT code for Val.



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  		FIG. 4. Expression of Go1{alpha} and Go2{alpha} transcripts in myometrium. Top: The cDNAs generated from nonpregnant (lanes 1, 2, 5, and 6) and pregnant (lanes 3, 4, 7, and 8) patients were amplified in the presence of primers specific for Go2{alpha} (lanes 1–4) and Go1{alpha} sequences (lanes 5–8) for 26 cycles. Reverse transcriptase was added (lanes 1, 3, 5, and 7) or omitted (lanes 2, 4, 6, and 8). RT-PCR products were stained with ethidium bromide after electrophoresis. Bottom: Splice pattern giving rise to Go2{alpha} and Go1{alpha} transcripts. Boxes represent exons, and interrupted lines, introns. Open boxes indicate the coding region while hatched boxes indicate 3'UTR.

When Go2{alpha} primers (Table 1; PCR-4) were used, a 357-bp cDNA fragment was obtained in myometrium from both nonpregnant and pregnant patients (Fig. 4, lanes 1 and 3). No amplification product was observed in the absence of reverse transcriptase (Fig. 4, lanes 2 and 4). Direct sequencing of the 357-bp cDNA fragment revealed the partial cDNA Go2{alpha} sequence as shown in Figure 5. The sequence of exon 7B (735–877) was the same as that of the Go{alpha} gene. In exon 8B (878–1065), two modifications were observed. First, codon 317, AAA, was changed to AAG in myometrium from a pregnant and a nonpregnant patient. The replacement of A by G does not generate a change in the encoded amino acid, lysine residue. Additionally, codon 321, AGC, coding for a serine, was found to be ACC, coding for a threonine (S321T) in nonpregnant and pregnant myometrium.



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  		FIG. 5. Partial nucleotide and deduced amino acid sequence of Go2{alpha} cDNA. Nucleotides are numbered on the left, amino acids on the right. Single-letter code for amino acids is used. The stop codon (***) is in bold face. The modified 317th and 321st codons are also in bold face and framed. Asterisks (*) noting nucleotides 951 and 962 and amino acid 321 indicate modifications observed. The site of intron 7 is shown by an arrow. The conserved G-4 (NKXD) and G-5 (TCATDT) motifs are underlined. The region (HXTCA) of high sequence identity among most {alpha} subunits crosses over G-5. The potential PTX modification site is indicated by a hexagon framing the cysteine, located four amino acids from the carboxyl terminus. EMBL Nucleotide Sequence data base Accession number Y18213.

Expression Levels of Go1{alpha} and Go2{alpha} Transcripts in Myometrium

Semiquantitative RT-PCR analysis of Go1{alpha} and Go2{alpha} transcripts was performed in myometrium from nonpregnant and pregnant patients. RT-PCR products corresponding to a Go{alpha} common region, and to Go1{alpha} and Go2{alpha} exon sequences, obtained by amplification with the previously defined number of cycles, were amplified with the previously defined number of cycles and visualized by ethidium-bromide (Fig. 6A). Relative intensities of the bands were calculated and normalized on a 100% scale. Results were expressed as the means obtained in myometrium from nonpregnant or pregnant patients (Fig. 6B). Levels of common Go{alpha}, Go1{alpha}, and Go2{alpha} RT-PCR products were significantly higher in myometrium from pregnant patients than in myometrium of nonpregnant patients (p < 0.01). Furthermore, it seems that there was a preferential increase in Go1{alpha} as compared to Go2{alpha} in pregnant myometrium. In contrast, no significant difference was observed between the two tissues for levels of ß2-microglobulin mRNA (Fig. 6, A and B) and 28S ribosomic RNA (data not shown), which served as external standards.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The strong expression of the 39-kDa Go{alpha} protein in myometrium from pregnant patients, shown here using three Go{alpha} antisera, is in agreement with the previously described increase in this protein as compared to the nonpregnant state [17]. Reactivity with a 40-kDa protein was observed in every myometrial sample. Cross-reactivity of Go{alpha} antisera with a Gi{alpha} protein can probably be discounted, since a substantial amount of the 40-kDa substrate, an abundant Gi2{alpha} protein contained in human platelets, was not detected by Go{alpha} antisera in platelet membranes [19, 27]. A Go{alpha} protein with a 40-kDa molecular mass has been reported in T tubules from rat skeletal muscle, rat myometrium, and human adipose tissue (discussed in [19]). The 40-kDa Go{alpha} immunoreactive protein may differ from the 39-kDa Go{alpha} either by its sequence or by differences in post-translational modifications. Indeed, Go{alpha} proteins undergo lipid additions such as myristylation and palmitoylation (discussed in [19]).

The data reported here, obtained with sequence-specific oligonucleotide primers and probes, show for the first time the expression of Go1{alpha} and Go2{alpha} transcripts in human myometrium and are consistent with the presence of Go{alpha} protein isoforms. In spite of the great homology between Go{alpha} and other G{alpha} proteins, we were able to isolate a Go{alpha} cDNA fragment common to both Go1{alpha} and Go2{alpha} by RT-PCR with external, and then nested, primers. This amplification product was identified by hydrolysis with a Go{alpha}-specific restriction enzyme, hybridization with an internal oligonucleotide, and sequencing. Go{alpha} was expressed in human myometrium and anterior pituitary gland while no expression was seen in a cell line of megakaryoblasts, progenitors of platelets. These results are in agreement with Go{alpha} immunoreactivity shown in rat anterior pituitary gland and its absence in human platelets [7, 27]. Indeed, Go{alpha} is expressed in varying amounts depending on the tissue. In the rat, it is abundantly expressed in neural tissues, less so in the pituitary gland (5% of Go{alpha} concentration in cerebrum), and to a lesser extent in some peripheral tissues including the uterus (0.5%), whereas no mRNA has been detected in liver, erythrocytes, leukocytes, and platelets [7]. Also, we did not observe Go{alpha} transcripts in uterine cervical myofibroblasts, which differ functionally from the myometrial smooth muscle cells and may be involved in the ripening process of the uterine cervix near term [18, 28].

Here, the presence of Go1{alpha} transcripts in human myometrium was demonstrated. Sequencing revealed the transcripts to be identical to the genomic Go1{alpha} sequence [10], while they differ from cDNA described in brain by Lavu et al. [16] by a synonymous modification of codon 339. This partial Go1{alpha} sequence was also found (unpublished data) in another muscular tissue, the smooth muscle from human placental villi vessels, which expresses a 40-kDa Go{alpha} protein at the same stage of pregnancy [19].

As far as we know, Go2{alpha} cDNA sequence in humans has not yet been described. We demonstrated the presence of Go2{alpha} transcripts in human myometrium by amplification of a specific cDNA region corresponding to the genomic Go2{alpha} region sequence. The partial exon 7B sequence obtained in myometrial cDNA was identical to that described in the genomic Go{alpha} sequence. Of the two nucleotide modifications observed in the exon 8B sequence of the cDNA, the 951st cDNA nucleotide (codon 317) was G in two cases out of three, while the corresponding genomic nucleotide has been described as A. This change did not generate amino-acid substitution. Tracings of the automatic cDNA sequencing showed an unambiguous G reading, although a low level of A was also observed (data not shown) suggesting a possible heterozygosity in these two patients. In contrast, the mutation of the third base in codon 317 was not found in fetal vessels from placental villi (unpublished results). These observations are in agreement with the single-base (A or G at the third position of codon 317 Lys) synonymous polymorphism, which has been previously described in the genomic Go{alpha} sequence [29]. The second modification we observed was seen in the 962nd cDNA nucleotide (codon 321). This nucleotide is G in the genomic sequence. In our sequencing experiments, this nucleotide was read as C in all myometrial samples. This modification was also observed in the muscular layer of placental stem villi vessels (unpublished data). This substitution generates the replacement of serine by threonine. This residue, found 3 amino acids before the conserved G-5 domain, appears to be located in the sixth ß chain of the GTPase domain [30]. Although Ser and Thr share similar polarity and neutrality, the addition of a methyl group in Thr may modify GTPase function. Similar mutations between neutral and polar amino acids have also been recently described [31]. A glycine-to-serine mutation (G184S) in the "Switch I" region of the Go{alpha} subunit gene prevents Go{alpha} protein deactivation, by causing insensitivity to a RGS (regulator of G protein signalling) protein [31].

High levels of Go{alpha} protein and transcripts were seen in human myometrium at the end of pregnancy. Very little is known about the mechanisms that determine cellular levels of Go{alpha} proteins and transcripts. Transient expression assays using deletion constructs of the mouse Go{alpha} gene (5' flanking sequence and 5' UTR) suggest that both transcriptional and post-transcriptional mechanisms are involved in regulating the expression of Go{alpha} in vivo [32]. Cyclic AMP may be among the intracellular factors that regulate transcription, since the 5' flanking region of the Go{alpha} gene contains a cAMP-responsive element [10, 32]. Furthermore, in neuroblastoma-glioma cells, the Go{alpha} level was elevated in the course of differentiation induced by dibutyryl-cAMP (reported in [32]). These data are compatible with a Go role in differentiation as shown in developing mouse brain [13] and cellular growth. Indeed, it appears that continuously active Go{alpha} can stimulate NIH-3T3 cell proliferation and transformation [33].

To elucidate the role of Go in a living animal, Go{alpha}-deficient mice were generated by targeted disruption of the mouse Go{alpha} gene and homologous recombination [34]. Go{alpha}-/- mice showed multiple neurological abnormalities and alterations in the regulation of Ca2+ currents in the heart [34] and died at a very early postnatal age.

In human myometrium, the transition from a relatively quiescent to an efficient contractile state at the end of pregnancy results from a shift in the systems that maintain the relaxed condition of the uterus. Stimulation of ß-adrenergic receptors has been shown to activate adenylate cyclase, leading to cAMP formation, which in turn contributes to the relatively quiescent state established throughout pregnancy. Disappearance of the adenylate cyclase stimulation by ß-adrenergic agents [35] and prostaglandins (PGE1, PGE2, and PGI2) (reviewed in [36]) has been observed at term, despite a high level of Gs{alpha} at the end of pregnancy [37]. Inhibition of adenylate cyclase activity mediated by {alpha}2-adrenergic receptors has also been reported at term [6]. Furthermore, it has been demonstrated that the coupling of {alpha}1-adrenoreceptors, endothelin, and prostaglandin (PGF2{alpha} and, to a lesser extent, PGE2) receptors to phospholipase C (PLC) results in phospho-inositide breakdown and inositol-triphosphate formation (reviewed in [36]). Inositol-triphosphate causes an increase in intracellular calcium and, subsequently, uterine contractility. Although the role of Go protein in myometrium is unknown, it has been shown in other systems to inhibit adenylate cyclase (as for example in transfected JEG-3 cells [38]) or to activate PLC as seen in Xenopus oocytes [39].

Additionally, in pregnant myometrium it seems that there is a preferential increase in Go1{alpha} as compared to Go2{alpha} mRNAs. This raises the question of whether different roles may be played by the two isoforms. Alternatively, the two isoforms may have the same function, the inhibition of calcium currents, but may be assigned to different receptors, as seen in rat pituitary GH3 cells [40].

In summary, we identified transcripts of the Go{alpha} gene in myometrium from pregnant and nonpregnant patients. Partial cDNA sequences of both Go1{alpha} and Go2{alpha} mRNA variants were described. The Go2{alpha} cDNA sequence differed from the Go{alpha} gene by two nucleotides in exon 8B. An increase in the expression of these two mRNA variants has been shown at the end of pregnancy and correlates with the increase of the two immunoreactive protein isoforms. Although there is not always a correlation between the level of proteins and their functions, we suggest that the significant amounts of Go{alpha} transcripts and proteins observed in human myometrium indicate a probable role in the very complex signaling pathways that contribute to physiological processes seen at the end of pregnancy.


   ACKNOWLEDGMENTS
 
We are very grateful to J. Doly (CNRS, UPR. 37, Paris), who offered support throughout our Go{alpha} research, and to C. Mehats and M.J. Leroy, who collaborated and provided their priceless assistance. We further acknowledge Professors J. Chavinié and M. Tournaire (Saint Vincent-de-Paul Hospital, Paris) and G. Grangé (Department of Obstetrics and Gynecology at Cochin-Port-Royal Hospital, Paris) for supplying myometrial samples and providing clinical information. Our great appreciation goes to B. Rouot (INSERM, U. 431, Montpellier, France) for the gift of Go{alpha} antibodies. We thank Y. de Keyser (Groupe d'Etude en Physiopathologie Endocrinienne, Institut Cochin de Génétique Moléculaire, Paris) and M.T. Mitjavila (INSERM, U. 362, Villejuif, France) for providing us with total RNAs from human normal adult pituitary gland and from the DAMI megakaryoblastic cell line, respectively; E. Dallot for preparing RNA from uterine cervical myofibroblasts; and C. Cruaud, G. Gapay (Genethon Laboratory, Evry, France), and J. Chelly (INSERM, U. 129, Paris) for helpful advice and sequencing. We also thank S. Allman for reviewing the English text and M. Verger for secretarial assistance.


   FOOTNOTES
 
1 Correspondence: Paulette Duc-Goiran, U. 361, Pavillon Baudelocque, 123, Bld de Port-Royal, 75014 Paris, France. FAX: 331 43 26 44 08; u361{at}cochin.inserm.fr Back

Accepted: January 27, 1999.

Received: December 29, 1998.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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