Mol. Hum. Reprod. Advance Access originally published online on January 19, 2008
Molecular Human Reproduction 2008 14(2):85-96; doi:10.1093/molehr/gam084
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Cloning, expression and immunolocalization of 
1-adrenoceptor in different tissues from rhesus monkey and human male reproductive tract
1Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua 3 de Maio 100, INFAR, Vila Clementino, São Paulo 04044-020, Brazil 2Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, NC 27599-7500, USA 3 Laboratories for Reproductive Biology, University of North Carolina at Chapel Hill, NC 27599-7500, USA
4 Correspondence address. Tel/Fax: +55-11-5576-4448; E-mail: avellar{at}farm.epm.br
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Abstract |
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This study reports the genomic organization of the rhesus
1A-adrenoceptor gene (ADRA1A). Full-length cloning of rhesus ADRA1A splice variants was achieved by combining PCR screening of a seminal vesicle cDNA library and 5'-RACE assays with total RNA from seminal vesicle. The classical ADRA1A mRNA (ADRA1A_v1) and six full-length ADRA1A splice variants were identified representing transcripts that code for functional (ADRA1A_v1, ADRA1A_v2a, ADRA1A_v3a, ADRA1A_v3d, ADRA1A_v3e) and truncated (ADRA1A_v2c and ADRA1A_v3c) receptor isoforms. Comparative analysis of the deduced amino acid sequence indicated that rhesus ADRA1A_i1 isoform (corresponding to the ADRA1A_v1 transcript) shares high identity to the amino acid sequence present in the classical 1A-adrenoceptor from human and other mammalian species. Partial nucleotide sequences for rhesus 1B-(ADRA1B) and 1D-adrenoceptor (ADRA1D) transcripts were also characterized. RT-PCR studies indicated differential distribution of all ADRA1A-related splice variants as well as ADRA1B and ADRA1D mRNAs, in tissues from rhesus and human male reproductive tract. Immunohistochemistry revealed 1A-adrenoceptor (ADRA1A_i1) immunostaining in smooth muscle cells and epithelial cells of rhesus efferent ductules, epididymis and seminal vesicle. Taken together the present results demonstrate that the complexity of the splicing mechanisms involved in the regulation of the ADRA1A gene is not restricted to human and is a common characteristic among Old World monkeys.
Key words:
1-adrenoceptor/human/male reproductive tract/rhesus monkey/splice variants
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1-adrenoceptors are members of the superfamily of G protein-coupled receptors that share a common structure with seven hydrophobic transmembrane domains (TM I–VII). 1-adrenoceptors bind the endogenous catecholamines epinephrine and norephinephrine and thereby mediate responses of the sympathetic nervous system such as smooth muscle contraction, myocardial inotropism and hepatic glucose metabolism (Lomasney et al., 1986; Minneman et al., 1988; Hieble et al., 1995; Graham et al., 1996). Stimulation of 1-adrenoceptors predominantly activates Gq/, resulting in hydrolysis of membrane phospholipids via phospholipase C-β and the generation of second messengers including inositol triphosphate and diacylglycerol, which mobilize intracellular calcium and activate protein kinase C, respectively (Graham et al., 1996; Michelotti et al., 2000).
Three 1-adrenoceptor subtypes (1A, 1B and 1D), corresponding to three different transcripts, have been cloned and pharmacologically characterized in several tissues and species, including human (Langer, 1999; Zhong and Minneman, 1999, Varma and Deng, 2000). All these three receptor subtypes show high affinity to the non-specific 1-adrenoceptor antagonist, prazosin (McGrath, 1982; Ruffolo, 1985). Pharmacological functional studies have indicated the existence of a fourth subtype (1L-adrenoceptor), which exhibits lower affinity to prazosin and has been reported to be involved with smooth muscle contraction of different human, rabbit and dog tissues (Muramatsu et al., 1994; Ford et al., 1996; Testa et al., 1996; Fukasawa et al., 1998, Hiraizumi-Hiraoka et al., 2004). There is also evidence suggesting that 1L-adrenoceptor may represent the low-affinity state of the 1A-adrenoceptor, and not a distinct receptor subtype (Ford et al., 1997). In the present work, the official IUPHAR 1-adrenoceptor subtype nomenclature (Foord et al., 2005) is used throughout, and the guidelines of the Human Gene Nomenclature Committee (HCGN, http://www.genenames.org) are used for naming 1-adrenoceptor transcripts and their corresponding isoforms (see Table I for reference).
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1A-, 1B- and 1D-adrenoceptor genes, denoted ADRA1A, ADRA1B and ADRA1D (Table I), are located on human chromosomes 8, 5 and 20 (Ramarao et al., 1992; Razik et al., 1997) and encode wild-type protein products of 466, 515 and 561 amino acids, respectively (Yang-Feng et al., 1990, 1994; Schwinn and Price, 1999). In addition to tissue- and species-specific patterns of 1-adrenoceptor expression (Rokosh et al., 1994; Hieble et al., 1995; Rudner et al., 1999; Silva et al., 1999; Queiróz et al., 2002; Errasti et al., 2003; Mendes et al., 2004; Scarparo et al., 2004), naturally occurring polymorphisms in the human ADRA1A gene have been shown to modulate sympathetically mediated physiological responses (Lei et al., 2005; Schaak et al., 2007). All three 1-adrenoceptor genes present a similar structure consisting of at least two exons separated by a large intron. One exon contains the nucleotide sequence that codes for the N-terminal region through the TM VI of the receptor, whereas the other exon encodes the third extracellular loop, TM VII and all (or most) of the C-terminal region (Ramarao et al., 1992; Esbenshade et al., 1995). Unlike ADRA1B and ADRA1D mRNA subtypes, which are expressed as a unique receptor form, the ADRA1A gene presents additional introns and several ADRA1A transcript variants have been isolated from human (Table I; Hirasawa et al., 1995; Chang et al., 1998; Cogé et al., 1999), rabbit (Suzuki et al., 2000) and guinea-pig (González-Espinosa et al., 2001) as a consequence of alternative splicing mechanisms. At least 11 different ADRA1A splice variants have been reported in humans (Table I). Four of these variants (wild-type ADRA1A_v1, ADRA1A_v2a, ADRA1A_v3a and ADRA1A_v4), differing in length and sequence of the C-terminal region, are functional proteins and indistinguishable from the classical wild-type 1A-adrenoceptor (ADRA1A_i1) with regard to their pharmacological profiles and abilities to mediate norepinephrine-induced [Ca2+]i release (Hirasawa et al., 1995; Chang et al., 1998; Cogé et al., 1999; Hawrylyshyn et al., 2004). It is suggested that the proteins coded by these ADRA1A splice variants might differ with regard to G-protein coupling specificity and down-regulation mechanisms (Hirasawa et al., 1995; Chang et al., 1998; Price et al., 2002). The other seven human splice variants (ADRA1A_v2b, ADRA1A_v2c, ADRA1A_v3b, ADRA1A_v3c, ADRA1A_v5a, ADRA1A_v5b and ADRA1A_v6) also differ in length and sequence of the C-terminal region, but encode for non-functional truncated isoforms that lack TM VII and are incapable of ligand binding and activation of signal transduction (Table I; Chang et al., 1998; Cogé et al., 1999; Hawrylyshyn et al., 2004). In spite of the high number of ADRA1A splice variants, the original wild-type ADRA1A_v1 transcript is the predominant variant in human prostate and other non-reproductive tissues such as heart, liver and pituitary (Hirasawa et al., 1995; Cogé et al., 1999; Schwinn and Price, 1999). It has been suggested that endogenous wild-type 1A-adrenoceptor (ADRA1A_i1) may be regulated by the presence of truncated receptors since their over-expression decreases plasma membrane and intracellular [3H]-prazosin binding sites for the wild-type 1A-adrenoceptor, possibly by affecting trafficking and membrane localization of these receptors (Cogé et al., 1999). The physiological, functional and developmental significance of these splice variants in human and other species remains to be determined.
In order to gain insight into the organization and regulation of the ADRA1A gene in primates, we have cloned and characterized the classical ADRA1A mRNA (ADRA1A_v1) and six different ADRA1A splice variants in rhesus monkey seminal vesicle. The distribution of all these ADRA1A splice variants, as well as ADRA1B and ADRA1D transcripts, in different tissues from rhesus male reproductive tract (testis, epididymis, seminal vesicle and prostate), was also analysed in comparison with human. Furthermore, the presence of the wild-type 1A-adrenoceptor was investigated by immunohistochemistry in rhesus efferent ductules, seminal vesicle and epidydimis. The present study demonstrates that the complexity of splicing mechanisms involved in the regulation of the ADRA1A gene may be a common characteristic among Old World monkeys.
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Tissues
Human and rhesus monkey (Macaca mulatta) testis, epididymis, seminal vesicle and prostate were made available by Dr James L. Mohler, Department of Urology/Surgery, and by the Tissue Procurement Core Facility of the Lineberger Comprehensive Cancer Center, University of North Carolina (Chapell Hill, NC, USA). Human tissues were not accompanied by identifying information and cannot be traced to the donor, and were obtained with informed consent from three patients ranging in age from 56 to 83 years. Rhesus tissues were from four animals of age 10–12 years, with proven breeding history (Covance Research Products Inc., Alice, TX, USA). All procedures were approved by the local Ethics Committee (process number 0913/03).
RNA isolation
Total RNA from human and rhesus monkey testis, epididymis, seminal vesicle and prostate was isolated with Trizol (Gibco–BRL, Gaithersburg, MD, USA), according to the manufacturer's instructions. RNA samples were then quantified using a spectrophotometer at 260 nm/280 nm and stored at –75°C for later use.
Cloning and characterization of full-length rhesus ADRA1A splice variants
Full-length cloning of rhesus ADRA1A splice variants was achieved by combining PCR screening of a directional rhesus seminal vesicle cDNA library and rapid amplification of 5'-cDNA ends (RACE method) with total RNA from seminal vesicle. Primers used for these studies were designed based on genomic and cDNA sequences from human ADRA1A gene (Table II).
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PCR screening was performed in a directional cDNA library from adult rhesus monkey seminal vesicle [poly (A)+] RNA in
ZAP II (Stratagene, Cedar Creek, TX, USA) previously obtained in our laboratory (Avellar et al., 2004). PCR amplifications were performed with an aliquot of the cDNA library (5 µl) in a final volume of 12.5 µl containing PCR buffer in the presence of 1.5 mM MgCl2, 0.2 mM dNTPs, 2 U Taq Polymerase (Gibco–BRL) and 0.4 µM of each sense and antisense primers. Pairs of primers were based on published human cDNA sequences and used to amplify rhesus ADRA1A_v1(AHF1/AHR1), ADRA1A_v2 (AHF1/AHR2) and ADRA1A_v3-related transcripts (AHF1/AHR3) (Table II). An initial cycle of 9 min at 94°C followed by 15 cycles of 1 min at 94°C, 2 min at 55°C and 2 min at 72°C were performed in a Perkin-Elmer 2400 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). After a final extension of 8 min at 72°C, the MgCl2 concentration of the samples was increased to 4 mM and the concentration of the other reagents maintained the same as above in a final volume of 25 µl. Samples were then submitted to further 25 cycles as described above. DNA samples (18 µl) were loaded onto agarose gels (1.8%) containing ethidium bromide (0.5 µg/ml). PCR products were visualized with fluorescent illumination and photographed. Amplicons were extracted, purified from the gel with Sephaglas Band Prep kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and subcloned into a pCRII vector using a TOPO TA Cloning kit (Invitrogen, San Diego, CA, USA). Faint DNA products observed during gel analysis were directly subcloned into the pCRII vector using aliquots (1 µl) of the respective PCR sample. Clones containing inserts were sequenced with an ABI PRISMTM 377 automated sequencer (Applied Biosystems) and BigDyeTM Terminator Sequencing kit (Applied Biosystems) at the DNA sequencing facility located at INFAR, Universidade Federal de São Paulo, Escola Paulista de Medicina, Brazil. Subsequent 5'-rapid amplification of cDNA ends (RACE) was performed to obtain the full-length ADRA1A clones, using the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) following manufacturers protocol. Briefly, oligo(dT)-primed cDNA was synthesized from 5 µg of total RNA from rhesus seminal vesicle. After synthesis of the second strand cDNA, the sample was ligated to Marathon cDNA adaptor (2 µM) in the presence of T4 DNA ligase (1 U/µl). Adaptor-ligated double-stranded cDNA (1 µl) was then diluted in Tricine–EDTA buffer (250 µl), heated at 94°C for 2 min and kept at –20°C until use. RACE PCR was performed in a final volume of 25 µl containing Advantage buffer and Advantage 2 polymerase (Clontech) in the presence of an adaptor primer (AP1, 5' CCA TCC TAA TAC GAC TCA CTA TAG GGC 3', Clontech) and one ADRA1A specific primer for amplification of 5'-end (reverse primers AHR1 and AHR1133, Table II). PCR conditions were based on the recommendations of the manufacturer. When cycling was completed, RACE DNA products were extracted from the gel, purified, subcloned into pCRII vector and sequenced as described above.
RT-PCR for detection of 1-adrenoceptor mRNA subtypes in human and rhesus male reproductive tract
The presence of ADRA1A splice variants in different tissues from human and rhesus monkey male reproductive tract (testis, epididymis, seminal vesicle and prostate) was analysed by RT-PCR. The distribution of the three different 1-adrenoceptor transcript subtypes (ADRA1A, ADRA1B and ADRA1D) was also evaluated in the same experimental samples. In this set of experiments, the partial nucleotide and predicted amino acid sequences for rhesus ADRA1B and ADRA1D were deduced.
RT-PCR amplification was performed using ThermoScriptTM RT-PCR System according to manufacturer’s instructions (Gibco–BRL). Reverse transcription of total RNA (5 µg) using oligo(dT)12–18 (2.5 µM) was performed for 1 h at 55°C. Routinely, reactions in the absence of reverse transcriptase were used as a negative control. PCRs with human and rhesus cDNAs were performed with specific pairs of primers for the amplification of ADRA1A (AHF497/AHR1133), ADRA1B (BHF256/BHR1099), ADRA1D (DHF616/DHR1155), ADRA1A_v1 (AHF1/AHR1), ADRA1A_v2 (AHF1/AHR2) and ADRA1A_v3-related transcripts (AHF1/AHR3) (Table II). Amplification of glyceraldehydes 3-phosphate dehydrogenase (GAPDH) mRNA was used as internal control. Primers used were GAPDH-F (5' CGG GAA GCT TGT GAT CAA TGG 3') and GAPDH-R (5' GGC AGT GAT GCC ATG GAC TG 3') (Scofield et al., 1995).
The resulting cDNA (2 µl) was amplified by PCR in a final volume of 25 µl containing PCR buffer (20 mM Tris–HCl, pH 8.4, 50 mM KCl) in the presence of 1.5 mM MgCl2 (3 mM for the amplification of ADRA1A transcript), 0.2 mM dNTPs, 2 U Taq polymerase (Gibco–BRL) and 0.4 µM of each sense and antisense primers. Amplification of ADRA1D transcript was performed with PCR Enhancer Buffer (Invitrogen), according to manufacturer’s instructions. PCR amplifications of ADRA1A and ADRA1B transcripts consisted of an initial cycle of 1 min at 94°C, followed by 35 cycles of 30 s at 94°C, 45 s at 62°C (or 65°C for ADRA1B) and 1 min at 72°C and a final extension of 3 min at 72°C. PCR amplifications of ADRA1D transcripts consisted of an initial cycle of 2 min at 95°C, followed by 30 cycles of 45 s at 95°C, 30 s at 65°C and 1 min at 72°C and a final extension of 2 min at 72°C. DNA products were extracted from the gel, purified, subcloned into pCRII vector and sequenced, as described above.
Rhesus 1-adrenoceptor nucleotide and protein sequence analysis
Nucleotide and amino acid sequences were predicted and aligned using the software BioEdit Sequence Alignment Editor (Hall, 1999). Rhesus ADRA1A introns and exons were estimated based on nucleotide alignment with human ADRA1A gene located at chromosome 8p23 (GenBank accession number AC134395
[GenBank]
). The identity of rhesus ADRA1A splice variants, ADRA1B and ADRA1D transcript sequences was compared with sequences available in the NCBI database for human and other species, using BLAST (Altschul et al., 1997). Predicted amino acid sequence for rhesus ADRA1A splice variants was aligned using ClustalW 1.8 software (http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) with corresponding human sequence and displayed using Boxshade 3.21 software (http://www.ch.embnet.org/software/BOX_form.html). The degree of amino acid identity of the 1-adrenoceptor subtypes from rhesus in comparison to human and other mammalian species was also analysed.
Immunohistochemical assays in tissues from rhesus male reproductive tract
Rhesus monkey efferent ductules, caput and cauda epididymis and seminal vesicles were used for immunohistochemical studies. Tissues were fixed promptly after excision in Bouin’s solution (75 ml saturated picric acid, 5 ml glacial acetic acid, 25 ml 37% formaldehyde) and embedded in paraffin as previously described (Hamil et al., 2000). Sections (6 µM) were deparaffinized, hydrated and then treated with 0.05 mg/ml trypsin for 5 min in phosphate buffer (PBS; 0.01 M, pH 7.5). After two 3 min washes with cold PBS, the sections were incubated with 2% normal rabbit serum (NRS) in PBS for 10 min at room temperature. Sections were then incubated overnight at 4°C with an affinity purified goat polyclonal antibody raised against the C-terminus of the human ADRA1A_i1 (amino acids 448–466; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), diluted in 1% NRS with 0.1% sodium azide (1:80). The sections were washed for 5 min in PBS, incubated for 10 min in PBS containing 2% NRS and then incubated for 1 h at room temperature with biotinylated secondary antibody (Santa Cruz Biotechnologies) diluted in PBS containing 1% NRS (1:200). Vectastain Standard ABC kit (avidin–biotin-complex horseradish peroxidase) (Vector Laboratories Inc., Burlingame, CA, USA) was then used to localize the biotinylated antibody. Following three 2 min washes in PBS, peroxidase activity was revealed by an incubation in Tris buffer (0.05 M, pH 7.6) containing 3,3'-diaminobenzidine (0.075%) and hydrogen peroxide (0.002%) for 10 min at room temperature. The reaction was stopped by 3 min wash in Tris buffer followed by another 3 min wash in PBS. The staining was enhanced by exposing the slides to 2% OsO4 vapors for 10 min. Sections were then counterstained with toluidine blue, dehydrated in graded alcohols, cleared with xylene and coverslipped with Permount (Fisher Scientific, USA). Control experiments included overnight pre-absorption (4°C) of the primary antibody with 10-fold excess of the corresponding blocking peptide (Santa Cruz Biotechnologies). All sections were viewed with a Nikon E800 microscope. Images were digitized using a CoolSNAP-Pro CCD digital camera and Image-Pro Express Software (Diagnostic Instruments Inc., Detroit, MI, USA). Images were processed using Adobe PhotoShop CS version 8.0 (Adobe Systems Inc., San Jose, CA, USA).
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Cloning of rhesus ADRA1A mRNA splice variants
PCR screening of a rhesus seminal vesicle directional cDNA library and 5'-RACE with total RNA from rhesus seminal vesicle were used to clone rhesus ADRA1A transcript variants. These experimental strategies were followed by subcloning and sequencing of the resulting PCR products. The entire coding sequences of seven rhesus ADRA1A splice variants were isolated, their nucleotide sequence was identified by automated DNA sequencing and their amino acid sequence was deduced. A graphical representation of the exons used by these message variants, in relation to the exon/intron structure from the human ADRA1A gene, is shown Fig. 1. Rhesus exons rE1, rE2/2a, rE3 and rE4/4a presented sequence identity to human exons E1, E5/5a, E7 and E8/8a, respectively (Fig. 1A). Rhesus exon rE3 diverged from the respective sequence in human exon E7 due to the existence of an insertion of 23 bp and three different 5' splice sites, suggesting species-specific regulation of ADRA1A mRNA splicing mechanisms (Fig. 1). On the basis of similarities with the human gene, rhesus splice variants were named ADRA1A_v1, ADRA1A_v2a, ADRA1A_v2c, ADRA1A_v3a, ADRA1A_v3c, ADRA1A_v3d and ADRA1A_v3e, all representing alternative splicing from a single gene (Fig. 1B). Computational methods (BLAST search at the Genome Sequencing Center at Baylor College of Medicine, http://www.hgsc.bcm.tmc.edu) located the genomic organization of ADRA1A gene in the rhesus monkey chromosome 8 (accession number NW_001122890).
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The nucleotide and deduced amino acid sequences of rhesus exons rE2a, rE3 and rE4/4a, which codes for rhesus ADRA1A variant-specific C-terminal regions, are shown in Fig. 2. Functional (ADRA1A_v1, ADRA1A_v2a, ADRA1A_v3a, ADRA1A_v3d and ADRA1A_v3e) and truncated (ADRA1A_v2c, ADRA1A_v3c) variants, corresponding to proteins ranging from 325 to 475 amino acids, were classified based on the presence and absence of the TM VII on their structure, respectively (Figs 1B and 2).
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Rhesus exon rE1 contained a common DNA sequence of 883 bp encoding amino acids 1–294 that correspond to N-terminus through the TM VI of the receptor present in all rhesus ADRA1A splice variants. Nucleotide differences (20/883, 97.7% identity) were observed when this sequence was compared to human exon E1. Only two of these nucleotide changes (nt 742 and 751) lead to changes in amino acid composition (M248V and A251T, respectively) located in the third intracellular loop (Fig. 3).
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Rhesus exon rE2 presented a DNA sequence of 385 bp encoding amino acids 295–423 that corresponds to distal third extracellular loop, TM VII and part of the C-terminal region present only in the functional variants (Figs 2 and 3). Nucleotide differences (11/385, 97.1% identity) were observed when compared with the respective sequence present in human exon E5. Five of these nucleotides (nt 901, 1046, 1099, 1121 and 1144) lead to the changes in amino acid composition located in the third extracellular loop (F301I) and initial C-terminal region (K349R, V367L, M374T and R382G, respectively) (Fig. 3).
Rhesus exon rE2a presented a DNA sequence of 131 bp encoding amino acids 424–466 that corresponds to the specific C-terminal region of the rhesus wild-type ADRA1A transcript (ADRA1A_v1) (Figs 2 and 3). When compared to human exon E5a, this DNA sequence contained only one nucleotide change (nt 1291), leading to one amino acid change (E431Q) that corresponds to a polymorphic residue in the human (Figs 2 and 3).
Rhesus exon rE3 contained DNA sequence encoding the specific C-terminal region of ADRA1A_v3-related sequences (Fig. 1). When compared to human exon E7, the nucleotide and amino acid sequence of each specific variant depended on the use of one of the three different splice sites located at the 5'end of rhesus exon rE3, suggesting species-specific regulation of ADRA1A pre-mRNA splicing mechanisms (Figs 1 and 2). Rhesus ADRA1A_v3a and ADRA1A_v3c transcripts use the same splice site described in human exon E7 (splice site 1, Fig. 1B) and show high similarity to human ADRA1A_v3a and ADRA1A_v3c transcripts, respectively. Besides the 23 bp insertion in their nucleotide sequence, located at position 1779 of the human ADRA1A_v3a variant, nucleotide differences (18/220, 91.8% identity) were observed when compared to the respective human transcripts. The rhesus ADRA1A_v3a contains the 23 bp DNA insertion in the non-coding region and only one of the different nucleotides (position 1284) lead to amino acid change (M428I) (Figs 2 and 3). In the rhesus ADRA1A_v3c mRNA, however, the 23 bp insertion introduced an earlier stop codon, leading to a shorter C-terminal region when compared to human ADRA1A_i3c protein sequence (Figs 2 and 3).
Rhesus ADRA1A_v3d and ADRA1A_v3e transcripts, using splice sites located 19 (splice site 2) and 342 bp (splice site 3) upstream the original site in the human exon E7, presented unique specific amino acid sequences in the C-terminal region when compared to human ADRA1A_i3-related sequences (Figs 1B, 2 and 3). Although present in the human genomic sequence, human transcripts derived from these two additional splice sites have not been reported to date. While the 23 bp insertion was present in the non-coding region of the rhesus ADRA1A_v3e transcript, this DNA sequence codes for the last 6 amino acids of the rhesus ADRA1A_i3d protein (Figs 2 and 3).
Rhesus exon rE4 contains a DNA sequence 158 bp long encoding amino acids 424-475 of the C-terminal region of the rhesus ADRA1A_i2a (Figs 2 and 3). When compared to human exon E8, only two nucleotide differences (nt 1280 and 1328) were observed, leading to two changes in amino acid composition (R427L and N443S, respectively) (Figs 2 and 3). A change in the reading frame of this 158 bp DNA sequence (rE4a) leads to a different amino acid composition present in the rhesus ADRA1A_i2c.
Rhesus exon rE4a presents a DNA sequence of 71 nucleotides encoding amino acids 295–363 of the rhesus ADRA1A_i2c with high similarity to human ADRA1A_i2c. When compared to the respective sequence in human exon E8a, nucleotide differences (3/229, 98.6% identity) led to only one change in amino acid composition (H359R) (Figs 2 and 3).
Comparative analysis of the deduced amino acid sequences indicated that rhesus ADRA1A_i1 (wild-type) shared high identity to the amino acid sequences present in the classical 1A-adrenoceptor from human (98.2%), guinea pig (94.4%), rabbit (94%), rat (92.7%), bovine (92.1%) and mouse (91.8%). The identity was higher in TM regions than in N- and C-terminal regions of the receptor protein.
Tissue distribution of ADRA1A splice variants
RT-PCR studies were performed to analyse the distribution of ADRA1A splice variants in human and rhesus testis, epididymis, seminal vesicle and prostate (Fig. 4A). No PCR products were detected when reverse transcriptase was omitted from the RT-PCRs, demonstrating that the amplified products were not from genomic DNA (data not shown). In humans, specific PCR products to ADRA1A_v1, ADRA1A_v2a, ADRA1A_v2c and ADRA1A_v3a splice variants were readily observed in all tissues analysed (Fig. 4A). In the rhesus, higher abundance of variants ADRA1A_v1, ADRA1A_v3a and ADRA1A_v3d were observed in comparison with low levels of ADRA1A_v2c and ADRA1A_v3e mRNAs (Fig. 4A). In this set of experiments, faint DNA products were also observed during gel analysis (not visible in Fig. 4A). Cloning and sequencing of these amplicons identified them as human ADRA1A_v2b, ADRA1A_v3b and ADRA1A_v3c and rhesus ADRA1A_v2a and ADRA1A_v3c variants.
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Expression and tissue distribution of ADRA1B and ADRA1D transcripts
RT-PCR studies were also performed to analyse the expression and tissue distribution of ADRA1B and ADRA1D, in comparison to ADRA1A transcripts, in tissues from the human and rhesus male reproductive tract (Fig. 4B). The sequence obtained for human ADRA1B mRNA (nt 292–1063) in the present study showed 100% identity with the available sequence in GenBank (accession number NM_000679 [GenBank] ), whereas sequence obtained for human ADRA1D mRNA (nt 628–1147) presented a silent A to G substitution at position 927 (accession number NM_000678 [GenBank] ). Cloning and sequencing of the rhesus ADRA1B amplicon, corresponding to TM regions I–VI, indicated a nucleotide identity of 98% when compared to human ADRA1B sequence. Multiple alignments indicated that the deduced amino acids for this rhesus ADRA1B amplicon were identical to the correspondent amino acid sequence present in the human
1B-adrenoceptor and highly conserved (
98% identity) when compared to 1B-adrenoceptor from other species (rabbit, hamster, rat and mouse). Computational methods (BLAST search at the Genome Sequencing Center at Baylor College of Medicine, http://www.hgsc.bcm.tmc.edu) located the genomic organization of ADRA1B gene in the rhesus monkey chromosome 6 (accession number NW_001120992).
Cloning and sequencing of the rhesus ADRA1D amplicon, corresponding to TM regions III–VI, presented a nucleotide identity of 97.3% when compared to human sequence (accession number NM_000678
[GenBank]
). Although most of these changes were silent, three nucleotide differences resulted in changes in the rhesus predicted amino acid sequence when compared to the human 1D-adrenoceptor (G317R, G320R and M321L). At least two of these changes (G317R and G320R) are due to polymorphic sites present in the human sequence. Multiple alignments also indicated that the deduced amino acids for this rhesus ADRA1D amplicon shared 94.2% and 91.9% identity to the correspondent amino acid sequence present in the rabbit and rat/mouse 1D-adrenoceptor, respectively.
Rhesus ADRA1B and ADRA1D nucleotide sequences obtained were submitted to GenBank (accession numbers AY13584 and EF195122 [GenBank] , respectively).
Immunohistochemical assays
Since rhesus ADRA1A_v1 transcript was systematically amplified in all tissues analysed, immunohistochemical assays with an antibody specific to ADRA1A_i1 isoform were performed in adult rhesus efferent ductules, caput and cauda epididymis and seminal vesicle. Immunostainings were detected in smooth muscle cells surrounding epididymal tubules and interstitial blood vessels, as well as epithelial cells in all tissues tested (Fig. 5). Epithelial cells presented a different pattern of immunostaining depending on the tissue analysed (Fig. 5A, C, E, G). In efferent ductules, ADRA1_i1 was localized in the apical region of epithelial cells (Fig. 5A). In caput epididymis, most epithelial cells presented a punctate immunostaining, whereas some cells presented a diffuse cytoplasmatic staining (Fig. 5C). In the cauda epididymis and in seminal vesicle, the presence of this adrenoceptor isoform was homogenously distributed throughout the cytoplasm of the epithelial cells (Figs 5E, G). The smooth muscle cells showed a slight punctate diffuse staining in the efferent ductules and caput epididymis (Fig. 5A, C), whereas cauda epididymis and seminal vesicle presented a more concentrated punctate staining at these cells (Fig. 5E, G). Immunostainings were significantly reduced when negative control experiments were performed with the primary antibody pre-absorbed with an excess of its respective blocking peptide (Fig. 5B, D, F, H).
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In the present work, seven full-length ADRA1A splice variants from rhesus monkey seminal vesicle were identified, indicating that the regulation of the ADRA1A gene by alternative splicing in primates is not restricted to human. Alternative splicing of the rhesus ADRA1A gene generated functional (ADRA1A_i1, ADRA1A_i2a, ADRA1A_i3a, ADRA1A_i3d and ADRA1A_i3e) and non-functional truncated isoforms (ADRA1A_i2c, ADRA1A_i3c) based on the presence and absence of the TM VII on their deduced protein structure, respectively. According to the results obtained, rhesus ADRA1A exons rE1, rE2/2a and rE4/4a presented high similarity to human ADRA1A exons E1, E5/5a and E8/8a, respectively. Transcripts including exons E1a, E2, E3, E4 and E6 from the human gene were not found in rhesus. Cloning of rhesus-specific ADRA1A_v3-related mRNA variants indicated that the rhesus exon rE3 diverged from human exon E7 due to the presence of two additional splice sites, leading to the expression of species-specific transcripts. Differences in alternative splicing mechanisms between human and rhesus monkey have been described for several genes and involve exon skipping (Bernard et al., 2004), use of alternative 3'splice sites (Martini et al., 1997; Rogers et al., 2004), use of species-specific exons (Martini et al., 1997, Miller et al., 2001, Avellar et al., 2004), as well as mutually exclusive events (Miller and Zeller, 1997; Hershberger et al., 2001; Avellar et al., 2004). Little is known, however, about the regulation of species-specific alternative splicing and its molecular determinants (Graveley, 2001; Xu et al., 2002; Maniatis and Reed, 2002; Ast, 2004).
Analysis of the deduced amino acid sequences indicates that rhesus ADRA1A isoforms contains all the ‘hallmark' residues that are highly conserved in the receptor family. For example, it contains two cysteine residues (C99 and C176) for the proposed dissulfide bond between the second and the third extracellular loops (Graham et al., 1996) and one cysteine residue (C345) at the C-terminal region, a potential substrate for palmitoylation (Karnik et al., 1993). Binding and signaling characteristics of the predicted amino acids from rhesus functional ADRA1A isoforms are probably identical to human, since the amino acid residues comprising the endogenous agonist (noradrenaline) binding site of the human 1A-adrenoceptor, including D106, S188, S192, F288 and F289 (Hwa and Perez, 1996; Porter et al., 1996; Perez et al., 1998) are also present in rhesus. In fact, competition binding studies using 1A-adrenoceptor and 1B-adrenoceptor selective antagonists in the liver of rhesus monkeys indicated a similar pharmacological profile of these adrenoceptors to human 1-adrenoceptors (García-Sáinz et al., 1996).
In the rhesus, the predicted number of putative phosphorylation sites differs between ADRA1A_i1 and ADRA1A_i2a, ADRA1A_i3a, ADRA1A_i3d and ADRA1A_i3e isoforms, suggesting that desensitizing mechanisms may differ among rhesus ADRA1A isoforms. However, deletion of the C-terminal intracellular region of human 1A-adrenoceptor had no effect either in the internalization rate or in the desensitization induced by agonist exposure or by G protein-coupled receptor kinase (GRK) over-expression (Price et al., 2002), suggesting that desensitization of 1A-adrenoceptor does not involve the C-terminal region. Alternatively, it is possible that the differences in the C-terminal region of the functional human and rhesus ADRA1A isoforms lead to different interactions between the receptor and accessory proteins, since the C-terminal tail of GPCRs is reported to interact with numerous proteins (Bockaert et al., 2004), generating networks involved in the trafficking, signaling and allosteric regulation of GPCRs.
1-adrenoceptors are widely distributed in tissues from rat and human and generally different subtypes are present in the same tissue (Rokosh et al., 1994; Silva et al., 1999; Queiróz et al., 2002; Errasti et al., 2003). In humans, mRNA for ADRA1A splice variants coding for functional and truncated receptor isoforms have been detected simultaneously in different tissues (e.g. liver, heart, brain, lung and prostate) with expression levels varying considerably for each variant (Schwinn et al., 1995; Hirasawa et al., 1995; Tseng-Crank et al., 1995; Chang et al., 1998; Cogé et al., 1999). In the present study, RT-PCR assays indicated that 1-adrenoceptor transcripts (ADRA1A, ADRA1B and ADRA1D) are present in all human and rhesus male reproductive tract tissues tested (testis, epididymis, seminal vesicle and prostate). The human and rhesus ADRA1A_v1 transcript, corresponding to the classical 1A-adrenoceptor (ADRA1A_i1), was abundant in all tissues analysed, in contrast to the other variants, whose amplification was variable among the tissues. The results are in agreement with the literature, since RNase protection assays (RPA) and quantitative RT-PCR reveal that the original cloned wild-type ADRA1A_v1 transcript (85–95% of total ADRA1A mRNA) is the most abundant ADRA1A variant in human heart and prostate (Cogé et al., 1999; Schwinn and Price 1999; Price et al., 2002).
No information is available in the literature about the expression of ADRA1A isoforms in different tissues and cell types. Taking into consideration that the ADRA1A_v1 transcript seems to be the most abundant in human and rhesus monkeys male reproductive tract, immunohistochemistry was performed in rhesus efferent ductules, epididymis (caput and cauda) and seminal vesicle using an antibody against classical 1A-adrenoceptor (ADRA1A_i1). This antibody has been successfully used in immunohistochemical detection of 1A-adrenoceptor in human prostate (Walden et al., 1999), vascular smooth muscle in rats (Hrometz et al., 1999), in human peripheral blood lymphocytes (Tayebati et al., 2000), in Western blotting with protein extracts from different rat tissues (Shen et al., 2000) and cultured HEK-293 cells transfected with recombinant human ADRA1A_i1 (Vicentic et al., 2002), as well as in our laboratory in immunohistochemical studies with rat and human epididymis, vas deferens and seminal vesicle (Queiróz et al., 2008), and in Western blotting with semi-purified membrane preparations from rat caput and cauda epididymis (Queiróz et al., unpublished data).
In the rhesus, ADRA1A_i1 immunostaining was observed in smooth muscle and epithelial cells from efferent ductules, caput and cauda epididymis and seminal vesicle. The presence of positive staining in the smooth muscle layer confirms the involvement of ADRA1A_i1 in the contractile responses induced by catecholamines in these tissues, as previously reported for vas deferens, cauda epididymis, seminal vesicle and prostate from different species (Hib and Caldeyro-Barcia, 1974; Marshall et al., 1995; Pupo, 1998; Honner and Docherty, 1999; Silva et al., 1999; Chaturapanich et al., 2002; Queiróz et al., 2002; Mendes et al., 2004). A specific immunostaining was also observed in the intracellular compartments of epithelial cells from rhesus monkey tissues. Although there is evidence that 1A-adrenoceptor participates in electrolyte transport (Wong and Yeung, 1978; Leung et al., 1992; Chan et al., 1994) and protein processing (Ricker et al., 1996) in epididymal epithelial cells, the physiological function of this adrenoceptor in epithelial cells from efferent ductules is unknown. One hypothesis is that 1A-adrenoceptor is involved in the regulation of protein secretion and in the concentration of testicular fluid which occurs specifically in this organ (Hermo and Morales, 1984; Hermo et al., 1988).
The intracellular presence of 1A-adrenoceptor has been described in the literature both in cells transfected with recombinant 1-adrenoceptor subtypes and in primary cultures of prostatic smooth muscle cells (Hirasawa et al., 2001; Piascik and Perez, 2001; Toews et al., 2003). In COS-7 cells transfected with 1-adrenoceptor subtypes coupled to green fluorescent protein, the distribution of 1A-adrenoceptor was predominantly detected throughout the cytoplasm of the cell (with enhanced perinuclear fluorescence), whereas 1B-adrenoceptor was mainly distributed on the plasma membrane (Hirasawa et al., 1997; Sugawara et al., 2002). Recombinant 1A-adrenoceptor can also be internalized in the absence of agonist exposure, suggesting that this traffic may represent a mechanism in which an internal pool of receptors is maintained and recycled (Morris et al., 2004). The ability of the intracellular 1-adrenoceptor to bind competitively to the fluorescent antagonist BODIPY FL-prazosin (Daly et al., 1998; Mackenzie et al., 2000; Sugawara et al., 2002) also raises the possibility that these receptors are functional and may be involved in signal transduction. The physiological and functional role of the intracellular rhesus ADRA1A_i1 reported in the present work will need further investigation. Furthermore, selective antibodies will be important tools for confirming abundance, subcellular localization and possible cell populations presenting specific expression of each ADRA1A isoform in the human and rhesus monkey male reproductive tract.
Data from the literature indicate that 1A-adrenoceptor is the predominant subtype in tissues from the male reproductive tract from different species, both at mRNA and protein levels (Price et al., 1993; Pupo, 1998; Silva et al., 1999; Pupo et al., 1999; Zhong and Minneman, 1999; Queiróz et al., 2002; Mendes et al., 2004; Jurkiewicz et al., 2006). Furthermore, 1A-adrenoceptor is known to be involved in pathological conditions such as benign prostatic hyperplasia and cardiac hypertrophy (Forray et al., 1994; Marshall et al., 1995; Rokosh et al., 1996; Moriyama et al., 1999; Autelitano and Woodcock, 1998; Bishop, 2007). The regulation of the expression and function of ADRA1A isoforms in these pathologies, however, remains unknown. The results of the present study demonstrate that the complexity of the splicing mechanisms involved in the regulation of the ADRA1A gene may be a common characteristic between human and Old World monkeys. Thus, the study of ADRA1A splice variants in a species more closely related to human can provide new insights into the investigation of their roles in human health and disease.
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Supported partially by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants 03/10 457-6 and 02/10 854-2, Brazil), by the National Institute of Child Health and Human Development/National Institute of Health through cooperative agreement U54-HD35 041 as part of the specialized cooperative Centers Program in Reproduction and by the T.W. Fogarty International Center for Training and Research in Population and Health, USA D43TW/HD00 627 (subcontract UNIFESP/UNC 5-53 284). Fellowships supported by FAPESP (M.T.C.C.P. and D.B.C.Q.), Brazil. Researcher fellowship to M.C.W.A. supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.
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We thank Espedita Maria de Jesus dos Santos, Maria Damiana Silva, Sabrina Silva and Luciana Honda for technical assistance.
Author roles
M.T.C.C.P. conducted the experiments for the genomic characterization of the rhesus ADRA1A gene, full-length cloning of ADRA1A splice variants, partial cloning of ADRA1B and ADRA1D transcripts, RT-PCR studies in human and rhesus tissues and wrote majority of the manuscript. D.B.C.Q., P.P. and G.G. performed and analysed the immunohistochemical studies. M.F.M.L. and M.C.W.A. supervised and coordinated the work and the preparation of the manuscript. All authors read, commented upon and approved the final manuscript.
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Submitted on October 2, 2007; resubmitted on November 24, 2007; accepted on November 28, 2007.
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