Structural, genetic and pharmacological identity of the rat a,-adrenergic receptor subtype cA2-47 and its molecular characterization in rat adrenal, adrenocortical carcinoma and bovine retina
Krzysztof Wypijewski*, Teresa Duda and Rameshwar K. Sharma
The Unit of Regulatory and Molecular Biology, Pennsylvania College of Optometry, 1200 West Godfrey Avenue, Philadel- phia, PA 19141, USA
Received 23 August 1994; accepted 30 November 1994
Abstract
Subsequent to the first a2-adrenergic receptor (a2-AR) gene cloning of a,-C10 from human platelet, cloning of the first rodent a,-AR cDNA, cA2-47, was reported. Based on the structural and limited pharmacological comparison, it was concluded that the rodent receptor is a molecular and pharmacological analog of the human receptor, which is pharmacologically classified as the a2-AR. A later study slightly revised the structure of the human receptor. Thus, the precise structural comparison of the rat receptor to the human platelet receptor is no longer valid. Another rat a,-AR gene, RG20, was then cloned and was also found to be a structural analog of the human a,-C10. It, however, varied slightly from the a2A subtype pharmacology, but matched the newly defined a2p subtype pharmacology. It was, therefore, concluded that RG20 encodes the a2p subtype. The structural and pharmacological relationship of RG20 with cA2-47 is not known, although it has been tacitly assumed that both are the identical a2p receptor subtypes. The present study addresses this and other issues relating to the precise structural, genetic and pharmacological relationship of cA2-47 with the human platelet a,-C10 receptor, and also the localization of cA2-47 tran- script in certain rat tissues. The results show that the cA2-47 receptor shows a high degree of sequence identity to the a,-C10 receptor, yet important differences exist between them. The sequence identity of cA2-47 receptor to the RG20 receptor is al- most, but not quite complete. The cA2-47 gene is not present in the human and the human gene is not present in the rat; that cA2-47 receptor subtype is pharmacologically similar to the RG20 receptor subtype, both being of the a2p subtype. The cA2- 47 receptor transcript in addition to being found in the rat brain is present in the rat adrenal gland, testes, adrenocortical carci- noma and the bovine retina. (Mol Cell Biochem 144: 181-190, 1995)
Key words: 2p-adrenergic receptor, cA2-47 pharmacology, a2D subtype localization
Introduction
In the early eighties, radioligand binding studies formed the basis for a pharamacological concept that the catecholamine a2-adrenergic receptor (a2-AR) was heterogeneous [1-5]. This concept was brought to the biochemical level by the first complete purification and characterization of a rodent (rat)
receptor which was shown to be equivalent to the pharma- cologically defined a __ subtype [6]. Soon thereafter the hu- man platelet a -AR was purified [7], and direct biochemical comparison of the rat and the human receptors showed that although their structures were closely related, they were not completely identical [8]. Thus, the two slightly-varied bio- chemical forms of a __ subtypes, rat and human, were estab-
*Present address: A. Mickiewicz University, Department of Biopolymer Biochemistry, Poznan, Poland.
Address for offprints: R.K. Sharma, The Unit of Regulatory and Molecular Biology, Pennsylvania College of Optometry, 1200 West Godfrey Avenue, Philadelphia, Pennsylvania 19141, USA
lished. This biochemical concept of the multiplicity of a,-AR subtypes moved to the molecular level with the first cloning of the a,-C10 gene which encoded the pharmacologically- defined human platelet @2, subtype [9]. Thereafter, the por- cine [10] and the rat analog, cA2-47 [11], of this clone were described. Comparison of the deduced amino acid sequence (450 residues) corresponding to the cA2-47 receptor with that of the human platelet receptor showed 84% sequence iden- tity. Despite this high sequence identity, the cA2-47 cDNA probe, under high stringency, did not hybridize with the hu- man genomic DNA. This indicated that the rat receptor subtype was nonexistent in the human genome [11]. After this study, two important developments in the a -AR field oc- curred. First, the structure of the human platelet receptor subtype was slightly revised [10]. Second, a rat gene, termed RG20 [12], was cloned which encoded a protein that showed a very high (89%) amino acid sequence identity to that of the cloned human platelet @24 receptor subtype, but its pharma- cology was slightly different from the human and instead matched the pharmacology defined for the a2 receptor subtype present in bovine pineal gland [13] and rat sub- maxillary gland [14]. It was, therefore, concluded that RG20 encoded the a2p receptor subtype. Although it has been as- sumed that RG20 and cA2-47 are identical molecules [15, 16], the molecular and pharmacological relationship of RG20 with cA2-47 is not known.
The present study addresses this and other issues relating to the precise structural, genetic and pharmacological relation- ship of cA2-47 with the human platelet a,-C10 receptor, and also the localization of cA2-47 transcript in certain rat tissues. The results show: (a) the precise structural differences between cA2-47,a,-C10 and RG20 receptors; (b) that the cA2-47 gene is not present in the human and the human gene is not present in the rat; (c) that cA2-47 receptor subtype is pharmacologi- cally similar to the RG20 receptor subtype, both being of the Q2p subtype; and (d) that cA2-47 receptor transcript in addi- tion to being found in the rat brain is present in the rat adrenal gland, testes, adrenocortical carcinoma and the bovine retina.
Materials and methods
Materials
Rat a,-AR (cA2-47) was cloned from the rat brain cDNA li- brary [11], the human platelet a,-AR genomic clone was pro- vided by Dr. C. Venter. [3H]rauwolscine, [32P]dATP and [32P]dCTP were from Amersham and ICN; yohimbine, oxymetazoline, phentolamine, p-aminoclonidine, prazosin and rauwolscine were from Sigma; cell culture media were from Gibco-BRL; restriction and modifying enzymes were purchased from USB, BioLabs, Promega and Pharmacia.
DNA sequencing
Nucleotide sequencing of cA2-47 was performed by dideoxy chain termination method [17] on double stranded plasmid template, using Sequenase 2.0 and 7-deaza-dGTP (USB).
Southern hybridization
Rat and human genomic DNAs were restricted with Kpnl and Sacl at 37℃. The restricted fragments were separated by agarose gel electrophoresis and the gels were dried. The dried gels were prehybridized at 65℃ in a buffer consisting of 5x Denhardt’s, 3x SSC and 0.1% SDS for 2 h and then hybrid- ized to [32P]-dATP-random labeled Kpnl/Sacl fragment of cA2-47 [11] or [32P]-end-labeled oligonucleotide probe corre- sponding to the amino acids 340-349 of the human platelet a2-AR (hp-340). The hybridization buffer was identical to that used for prehybridization except it contained 0.5 million cpm/ml of labeled cA2-47 or 0.65 million cpm/ml of labeled- hp-340. After 18 h of hybridization, the gels were washed at 55°℃ for 1 h in 2x SSC plus 0.5% SDS (two times) followed by a final high stringency wash in 0.3x SSC, 0.1% SDS at 55°℃ for 40 min. Gels were autoradiographed at -70°C.
Northern hybridization
Poly(A+)RNA was isolated from rat brain using mRNA Iso- lation Kit (Invitrogen). The mRNA was heated for 20 min at 65°℃ in a denaturation buffer which was 5.85% formalde- hyde, 24.5% formamide, 3.5 mM EDTA, 17 mM MOPS [3- (N-morpholino)-propanesulfonic acid], 4.3 mM sodium acetate and 10 UM ATA (aurintricarboxylic acid). After the addition of 1/10th (vol/vol) of a sample buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 50% glycerol, 1 µM ATA), the mRNA was applied to 1.2% agarose gel containing 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA and 0.1 mM ATA and electrophoresed at 130 V for 2 h. The gel was dried and prehybridized at 64℃ for 2 h in 5x SSC, 5x Denhardt’s solution [1× Denhardt’s contains per 500 ml 0.1 g Ficoll 400, 0.1 g polyvinylpyrrolidone and 0.1 g bovine serum albumin (Pentax Fraction V)], and 0.1 mg/ml sheared salmon sperm DNA. The gel was then hybridized at 60°℃ for 20 h in a solution identical to the prehybridization buffer and containing 0.5 million cpm/ml of [32P]-labeled probe. A Kpnl/Sacl fragment of cA2-47 [11] was randomly labeled with [32P]-dATP and used as a hybridizing probe. After the hybridization was completed, the gel was washed in 0.1x SSC at room temperature (three times, each 3 min) followed by 2x SSC plus 0.5% SDS (sodium dodecyl sulphate) at 55℃ (two times, 20 min). The final wash was at 55℃ for 40 min in 0.5x SSC plus 0.1% SDS (medium stringency
conditions). XAR5 (Kodak) film was exposed to the gel at -70℃. Northern hybridizations under low or high stringency conditions were performed as described previously [11].
Reverse transcriptase PCR (RT-PCR) amplification
A) cDNA synthesis
4 µg of mRNA isolated from various tissues was employed individually for one reverse transcriptase reaction in a mix- ture containing 50 µM random 9-mers (Stratagene), 0.4 U/ ul AMV reverse transcriptase (USB), 2.5 mM each of dNTPs, 1 U/ul RNasin (Promega), 25 mM MgC12, 50 mM KCI, 10 mM Tris-HCI (pH 9 at 25℃), 0.1% Triton X-100. The reac- tion mixture was preincubated at 20℃ for 15 min, incubated at 42°℃ for 25 min and the reaction was stopped by heating at 99°℃ for 3 min.
B) PCR amplification of the a,-AR transcripts
The 190 bp fragment (nucleotides 1127-1316) of cA2-47 was amplified from cDNA (vide supra) using the following prim- ers: 5’-TGGCGGTGGTGATCGGCGTGTTG-3’ (sense) and 5’-TTCTTGGGGAAGGCGCGGCGGAGTC-3’ (antisense), in a reaction mixture of 50 mM KCI, 10 mM Tris-HCI (pH 9 at 25°℃), 0.1% Triton X-100, 2 mM MgCl2, 0.2 mM each of dNTP, 1 uM of each primer and 2.5 U of Taq DNA poly- merase; with a thermal profile of: 2 min at 94℃, 25 cycles of 1 min at 94℃ followed by 1 min at 60℃ and 1 min at 72℃; and finally 3 min at 72℃. Amplified-products (1/5th aliquot of the reaction mixtures) were electrophoresed on 2% agarose gels containing 0.5 µg/ml of ethidium bromide. The control reactions for each tissue were performed under iden- tical conditions except that in these cases 1 µg of RNase-free DNase-treated mRNA was used as a template.
Expression studies
COS-7 cells (simian virus 40-transformed African green monkey kidney cells) were transiently transfected with cA2- 47 cDNA or human a,-C10 DNA ligated into pSVL expres- sion vector using the calcium phosphate technique [18]. COS-7 cells transfected with pSVL vector alone were used as controls.
Receptor binding studies
60 h after transfection, cells were rinsed with phosphate- buffered saline, pH 7.5, scraped into membrane suspension buffer (50 mM Tris-HC1, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 30 µM phenylmethylsulfonyl fluoride), homog- enized, centrifuged for 15 min at 5,000 x g and washed in
the same buffer. The pellet represented the crude membranes. Binding assays were performed in a total volume of 250 ul incubation buffer (10 mM MgC12, 50 mM Tris-HC1, pH 7.5) containing 50-100 µg of membrane proteins. [3H]rauwol- scine (15 nM or 5 nM for the rat or the human receptor, respectively), with or without competing agonists or antago- nists, was added and allowed to incubate at 25℃ for 40 min. Incubation was terminated by diluting the mixture with 3 ml of incubation buffer followed by immediate filtration through Whatman GF/C glass filters followed by 3 washes of 3 ml of ice-cold buffer. Specific binding was calculated by subtract- ing the nonspecific radioactivity from the total radioactivity bound to the filters. Experiments were conducted in dupli- cate and repeated at least three times for reproducibility. The results were expressed as the mean value of one typical experiment.
Protein determination
The protein was determined using a kit marketed by Pierce with BSA as a standard [19].
Results
Structural comparison of rat cA2-47, human a,-C10 and rat RG20
The deduced amino acid sequences of cA2-47, RG20 [12] and human a,-C10 [9,10] are compared in Fig. 1. There is 90% overall sequence identity between cA2-47 receptor and the human platelet receptor. Previous comparison had shown 85% identity between them [11].
The third cytosolic loop is the region of lowest identity (82%); previous comparison had shown the identity of 72% [11]. These changes from rat to the human receptor are in 27 residues: C329→V; S240→A; A246→T; D247→E; G258→S; T261>P; A262→G; T233→A; R284→G; G288→T; E300→D; Q306 >P;G309→R; K310→R; A317→G; T320→A; K322→R; A340 >T; P 342→1; A34>T; S345>P; G346>A; S347 >A; Q349 >P; A354 >V; G356 >A; R368>L.
The structural identity between the two receptors is com- plete in the first and the second cytosolic loop, and in the cytosolic COOH-terminal; there are two changes at the ex- tracellular NH,-terminal from rat to the human receptor, 2 S .- >A and T2,→A; one change V104>T in the first extra- cellular loop; 4 changes, A> >G, Q18 >P, S18]>R and 23 K139→E in the second extracellular loop; and one change from P400→S in the third extracellular loop.
There are some minor changes from rat to the human receptor in the first (F46 >L); fourth (V 15>I, V15] >C); fifth
| Ist Transmembrane | ||||
|---|---|---|---|---|
| RAT CAD-47 | MGSLQPCAGN SSWNGTEAPG | GGTRATPYSL QVTLTLVCLA | GLEMLFTVFG | 50 |
| RAT RGZO | MGSLQPDAGN SSWNGTEAPG | GGTRATPYSL QVTLTLVCLA | GLLMLETVFG | |
| HUMAN C-10 | MGSLOPDAGN ASWNGTEAPG | GGARATPYSL QVTLTLVCLA | GLLMLLTVEG | |
| 2nd Transmembrane | ||||
| RAT CA2-47 | NVLVIIAVET SRALKAPQNL | FLVSLASADI LVATLVIPES | LANEVMGYWY | 100 |
| RAT RG20 | NVLVIIAVET SRALKAPONL | FLVSLASADI LVATLVIPES | LANEVMGYWY | |
| HUMAN C-10 | NVLVIIAVET SRALKAPONL | FLVSLASADI LVATEVIPES | LANEVMGYWY | |
| 3rd Transmembrane | ||||
| RAT CA2-47 | EGKVWCEIYL ALDVLECTSS | IVHLCAISLD RYWSITQAIE | YNLKRTPRRI | 150 |
| RAT RG20 | EGKVWCEIYL ALDVLECTSS | IVHLCAISLD RYWSITQAIE | YNLKRTPRRI | |
| HUMAN C-10 | FGRTWCETYL ALDVLECTSS | IVHLCAISLD RYWSITQAIE | YNLKRTPRRI | |
| 4th Transmembrane | 5th | |||
| RAT CA2-47 | KAIIVIVWVI SAVISÉPPLI | SIĘKKGAGGG QQPAEPSCKI | NDOKWYVISS | 200 |
| RAT RG20 | KAIIVTVWVI SAVISEPPLI | SIĘKKGAGGG QQPAEPSCKI | NDOKWYVISS | |
| HUMAN C-10 | KAIIITCWVI SAVISEPPLI | SIEKKGGGGG POPAEPRCEI | NDQKWYVISS | |
| Transmembrane | ||||
| RAT CA2-47 | SIGSEFAPCL IMILVYVRIY | QTAKARTRVP PSRRGPDACS | APPGGADERP | 250 |
| RAT RG20 | SIGSFFAPCL IMILVYVRIY | OLAKRRTRYP PSRRGPDACS | APPGUADRRE, | |
| HUMAN C-10 | CIGSEFAPCL IMILVYVRIY | QTAKRATRYP ESRAGEDAVA | APEGOTERRE | |
| RAT CA2-47 | NOLGPERGAG TAGAEREEUP | TOLNGAFGER APTRPROGDA | LOLEESSSSE | 300 |
| RAT RG20 | NAVGPERGAG TAGAEREPER | TOLNGAPGER AFTRERDGDA | LDLEESSSSE | |
| HUMAN C-10 | NOLGPERSAG DECAFREFER | TOLNGAEGEE APAGPROTDA | LOLEESSSSI | |
| RAT CA2-47 | HAERPOGPEK PERGPRAKGK | FKASOVKPGD SEPRRGEGAN | GUGASGSGOG | 350 |
| RAT RG20 | HAEPROGECK PERGPRAKGK | SKASOYKEGD SERRAGEGAA | GPGASGSGOG! | |
| HUMAN C-10 | HAERPEGER PERGPROKOK | ARABOVKPGD, SLERRGPGAT, | GIGTPAAGPG | |
| 6th Transmembrane | ||||
| RAT CA2-47 | BERAGOAKAS,KURGRONREK | RETEVLAVVI GVEVVCWEPE | FFTYTLIAVG | 400 |
| RAT RG20 | EERAGGAKAS RWRGRONREK | RETTVLAVVI GVEVVCWEPE | EFTYTLIAVG | |
| HUMAN C-10 | EERYGRAKAS PWRGRONEEK | RETĘVLAVVI GVFVVCWEPE | FETYTLTAVG | |
| 7th Transmembrane | ||||
| RAT CA2-47 | CPVPYQLENE E-WEGYCNSS | LNPVIYTIEN HDFRRAEKKI | LCRGDRKRIV | 150 |
| RAT RG20 | CPVPYQLENE FEWEGYCNSS | LNPVIYTIEN HDERRAFKKI | LCRGDRKRIV | |
| HUMAN C-10 | CSVPRILEKF FEWEGYCNSS | LNPVIYTIEN HDERRAEKKI | LCRGDRKRIV | |
(S201→C); sixth (1317→T); and seventh (Y 405→R, Q406 >T and N409→K) transmembrane domains. There is complete iden- 409 tity between the two receptors in the second and third transmembrane domains.
There are five structural changes, all located in the third cytoplasmic domain, between the rat proteins cA2-47 and RG20. These changes from cA2-47 to RG20 involve the residues G25>>A; L253 >V; R304 >P; P305 >R and P3 >R. 252 253 305 304
It is noted that resequencing of cA2-47 resulted in the following changes from its previously published structure [11]: R147 >P; H154→I; C155→V; H156 >T; C157→V; V158 >W; G264→A.
Southern-blot comparison of rat cA2-47 and human a,-C10
Using the rat-specific cA2-47 and the human-specific a,-C10 probe, it was previously concluded that the rat receptor gene does not exist in the human genome and the human receptor gene does not exist in the rat genome, indicating the inde- pendent nature of these receptor subtypes in the rat and the human [11]. To reverify the validity of this conclusion, the
lane 1
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cross-hybridization of the rat probe to the human DNA and of the human probe to the rat DNA was tested as described before [11]. The [32P]-labeled Kpnl/Sacl-restricted cA2-47 1800 bp probe did not hybridize to the (Kpnl/Sacl-restricted) human genomic DNA but hybridized to a single 1800 bp fragment of Kpnl/Sacl-restricted rat genomic DNA (Fig. 2: lanes 1 and 2), indicating the presence of cA2-47 gene in the rat and its absence in the human genome. To determine if the human gene encoding a2 -AR was present in the rat genome, the labeled oligonucleotide probe corresponding to the amino acid residues 340-349 (hp-340) of the human platelet o2A- AR gene was used. A single hybridization band at 5.3 kb was observed in the Kpnl/Sacl-restricted human genomic DNA, but there was no hybridization with the rat Kpnl/Sacl-re- stricted DNA (Fig. 2: lanes 3 and 4). It is thus concluded that the rat and the human receptors are encoded by distinct spe- cies-specific genes.
cA2-47 receptor mRNA expression in rat brain and other tissues
The first direct evidence of a surprisingly large range of mRNA sizes for any member of the a,-receptor family in rat was provided by northern-blot analysis using the cA2-47 cDNA probe [20]. Depending on the stringency conditions,
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4 to 1 transcripts were found in rat brain and the adrenal tumor. To scrutinize the existence of the real and the cA2-47- related transcript/s in rat brain, northern-blot analysis of poly(A+)RNA from rat brain was performed (Fig. 3). Hybri- dization under low stringency showed 4 different sized mRNAs, under medium stringency 2, and under high strin- gency 1 (between 4-4.5 kb) (Fig. 3). It is important to note that under high stringency, the human probe failed to hybrid- ize with any rat message (data not shown), as was also ob- served earlier [11]. Two important conclusions are drawn from these findings. One, the human a2-AR transcript, just like its gene, is not present in the rat. Two, 4-4.5 kb is the size of the cA2-47 transcript, as reported earlier [11].
To determine the expression of cA2-47 in various tissues, the reverse transcription of mRNA isolated from rat adrenal gland, the adrenocortical carcinoma, brain, testes and bovine retina, was performed, which was followed by amplification of the 190 bp-fragment corresponding to cA2-47 nucleotides 1127-1316 (amino acid residues 382-439), using the poly- merase chain reaction.
Figure 4A shows the RT-PCR-amplified mRNA product in the rat adrenal gland, the adrenocortical carcinoma, brain, testes and bovine retina. It is noteworthy that in each tissue the am- plified-product corresponded to a single, expected 190 bp-frag- ment. [Amplification of the 190 bp-fragment from rat genomic DNA constituted the positive control (Fig. 4A, lane ‘con- trol +’) and the negative control (Fig. 4A, lane ‘control -’)
molecular weight standard
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consisted of all reaction reagents, except that DNA template was not added.] The lack of signals in the control samples and the exclusive synthesis of the precisely-sized RT-PCR- generated cA2-47 cDNA fragment in each tissue attested to the validity and the specificity of the RT-PCR method in re- vealing the molecular existence of the cA2-47 190 bp- fragment in all of the tested tissues. [The control reaction for each of the indicated tissues (Fig. 4A, lanes ’-’) was per- formed using RNAse-free DNAse-treated mRNA instead of cDNA.] To further verify the authenticity of the PCR product, the 190 bp-fragment amplified from the genomic DNA was
0.1
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Specific [‘H]Rauwolscine binding
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restricted with the specific endonuclease, Banl, which re- sulted in the anticipated restricted-fragment sizes of 80 and 110 bp (Fig. 4B). It is, therefore, concluded that the mole-
cular subtype cA2-47 receptor is expressed in the rat adrenal gland, the adrenocortical carcinoma, brain, testes and bovine retina.
Specific [ H]Rauwolscine binding (% of total)
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Pharmacological identity of cA2-47 receptor
To determine and compare the ligand-binding characteristics of cA2-47 receptor with the human _ subtype, the two receptor proteins were expressed in COS-7 cells. Scatchard plot analysis indicates that the specific binding of [3H]rauwol- scine to cA2-47 and a,-C10 is linear with the respective cal- culated Ka values of 12.9 nM and 2.5 nM (Fig. 5A); the latter value is close enough to the published value of 1.7 nM [12]. This indicates that the rawolscine affinity for the cA2-47 receptor subtype is in excess of 5-fold lower than that of the
Specific [ H]Rauwolscine binding
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a2-C10 receptor, a conclusion supported by the competition binding studies in which rauwolscine, yohimbine and oxy- metazoline were used to displace the [3H]rauwolscine bound to cA2-47 anda,-C10 receptors (Figs. 5B-H; Table 1). Com- pared to a.2-C10 receptor, cA2-47 receptor showed nearly 7- fold lower affinity for rauwolscine and yohimbine and 9-fold lower affinity for oxymetazoline; and about 50% higher affinity for the a, -AR specific antagonist prazosin and for the a-AR antagonist phentolamine. In addition, compared to a2- C10, cA2-47 showed 2-fold lower affinity for epinephrine, the natural a2-AR ligand and equal affinity for p-amino-
| Ligand | Ki (nM) | ||
|---|---|---|---|
| cA2047 | C10 | RG20 | |
| p-Aminoclonidine | 150 | 150 | |
| Phentolamine | 10 | 21 | 6 |
| Prazosin | 2500 | 4000 | 1586 |
| Rauwolscine | 75 | 12 | 51 |
| Yohimbine | 90 | 15 | 61 |
| Epinephrine | 2500 | 1100 | 2100 |
| Oxymetazoline | 60 | 10 | 43 |
clonidine, an a -AR agonist. It is, therefore, concluded that based on most of these ligand binding characteristics, cA2- 47 receptor can be pharmacologically distinguished from the a,-C10 receptor.
When the above binding characteristics of cA2-47 receptor are compared with those of the available literature values of the RG20 receptor, the two receptor subtypes appear to be similar (Table 1). The minor differences between them may be attributable to the different expression systems; cA2-47 was expressed in COS-7 cells and RG20 in COS-1 cells [12], or human variability. It is, therefore, concluded that the two receptor subtypes, cA2-47 and RG20 are pharmacologically identical or very similar, both belonging to the a2p subtype.
Discussion
Through molecular cloning studies, the presence of three dis- tinct a2-AR subtype genes - termed a,-C10, a2-C2, a2-C4 according to their chromosomal localization - have been es- tablished in the human [9, 21-23]. These genes encode the pharmacologically defined a2-AR subtypes: 24, C2B and O2c [15]. This pharmacological classification of a,-AR subtypes is based on the ligand binding studies: The & __ subtype has high affinity for oxymetazoline and low affinity for prazosin; the Q2B subtype has low affinity for oxymetazoline and high af- finity for prazosine, and the &2 subtype has high affinity for rauwolscine and prazosin [24-26]. This precisely-defined subtype pharmacology, however, does not completely match the pharmacology of the a, subtype present in the bovine pin- eal gland [13] and the rat submaxillary gland [14]. This receptor subtype has now been termed as the &2p, which is almost similar to the a2, subtype pharmacology, except for its slightly higher prazosin affinity and moderate affinity for rauwolscine and yohimbine [13]. In the rat this pharmacologically-defined subtype is encoded by the gene, RG20 [12]. Because to date the existence of the a2p subtype has not been observable in the
human and of the a, subtype in the rat (or other rodents), this has raised an important biological and evolutionary question: Is the rat gene RG20 encoding a20 subtype, the rat homologue of the human a,-C10 gene encoding the a2 subtype?
This issue was partly resolved in a study which reported the cloning of an a,-AR, termed cA2-47, from rat brain [11]. This study concluded that cA2-47 is the rat homologue of the hu- man a,-C10, which encoded the rat a __ subtype. At that time the a2p subtype was not pharmacologically defined, so it was not possible to determine if a cA2-47 encodes the a2 subtype.
The present study shows that there is a high enough (90%) sequence identity of rat cA2-47 receptor to human a,-C10 receptor to consider that the human and the rat receptors are structural analogs. Despite this strong shared-sequence iden- tity, there is important sequence dissimilarity between the rat and the human to suggest that the two forms are independ- ent structural entities: (1) The nucleotide comparison of the human gene and the rat cDNA shows that there is no sequence similarity between them over the 177 bases of the 3’- noncoding sequence available for comparison and over the stretch of 210 nucleotides in the 5’-untranslated region where the identity is less that 50% [11]; (2) the region of lowest iden- tity is the third cytosolic loop, and this is the region which is supposed to functionally participate with the Gi-protein- mediated inhibition of the adenylate cyclase system; (3) un- der stringent hybridization conditions, the rat cDNA probe only recognizes a single mRNA in rat tissues, none is detected by the human probe; (4) most significantly, under stringent conditions the rat cDNA probe does not recognize the human gene and the human gene probe does not recognize the rat gene. It is thus concluded that the rat and human receptors possess significant important structural and genetic differ- ences and that each form is species-specific.
Ligand binding studies indicate that both cA2-47- and a,- C10-encoded receptors have low affinity for prazosin, a char- acteristic that distinguishes these receptors from the a2B and C2c subtypes. Buta2-C10- and cA2-47-encoded receptors can be segregated from each other on the basis of their binding affinity for rauwolscine, yohimbine and oxymetazoline. The affinity of the rat receptor for these drugs is 5-10-fold less than the human receptor. This binding-affinity characteris- tic of the rat receptor matches the pharmacological criteria defined for the &2p subtype, and furthermore, is compatible with its rank order of potency for competing ligands estab- lished for this subtype in rat salivary gland [14] and RG20- transfected cells [12]. It is thus concluded that cA2-47 encodes the a2p subtype.
Barring changes in the 5 amino acid residues, cA2-47 receptor shows complete structural identity to the RG20 pro- tein. All these changes are located in the third cytoplasmic loop of the proteins. Pharmacologically, the two receptors are virtually identical, and previous studies have established that RG20 is the molecular form of the pharmacologically-defined
a2p subtype. If indeed the structural integrity of cA2-47 is distinct from that of RG20, this would indicate that both of these molecular forms represent the same pharmacologically- defined @2p-AR subtype.
By northern-blot analysis, the specific presence of cA2- 47 mRNA was previously shown in rat brain [11]. In the present study, through RT-PCR studies it is shown that includ- ing rat brain, the cA2-47 molecular subtype is present in the rat adrenal gland, the adrenocortical carconoma, testes and bovine retina. Molecular nature of this (or RG20) subtype has also been recently characterized in the rat superior cervical ganglia [16] and a mouse analog of this gene encoding the Q2p subtype has been characterized [27]. In addition to these tissues, this subtype has been pharmacologically character- ized in rat submaxillary gland [14] and rat [28] and bovine pineal glands [13]. This indicates the presence of this subtype in a wide variety of tissues belonging to at least three animal species, rat, mouse and cow.
The independent genetic and pharmacological identity of C2D subtype strengthens the previous concept of four a2-AR subtypes: a24, a2B, C20, C2D; the middle two subtypes are re- spectively encoded by the human genes a2-C4 [21] and a2- C2 [22, 23] and by their rat counterparts RG10 [12, 29] and RNGa2 [30, 31]; the third subtype a24 is encoded by the human a2-C10 gene, but so far has no counterpart in the rat and in other rodent species; similarly, a2D subtype is encoded by rat cA2-47 (and RG20), but to date has no counterpart in human. If such is indeed the case, this raises an interesting evolutionary question: What is so special about the a2 and Q2p subtype genes that they evolved to be species-specific functionally independent identies, but this did not occur in the case of the a23 and azc subtypes?
In conclusion, this study has established that cA2-47 is a molecular form of the pharmacologically-defined a2p sub- type, that this molecular form does not exist in the human, and in addition to the rat brain, this molecular form is also present in the rat tissues of the adrenal gland, testes, the adrenocortical carcinoma, retina and also the bovine retina.
Acknowledgements
This work was supported by the grants from the National Institutes of Health (NS 23744, EY 10828) and the equipment grant from Pennsylvania Lions Eye Research Foundation.
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