Vol. 183, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1090-1096

March 31, 1992

Angiotensin H Type-1 Receptor Subtype cDNAs: Differential Tissue Expression and Hormonal Regulation Sham S. Kakar, Jeffrey C. Sellers, Daniel C. Devor, Lois C. Musgrove, and Jimmy D. Neill Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294 Received

February

i0,

1992

Summary: A rat angiotensin, type 1A (AT1A) receptor cDNA was cloned recently and shown to be a member of the 7-transmembrane, G-protein coupled family of receptors. Here, we report the cloning, sequencing, and expression of a previously unsuspected second form of the type 1 receptor (AT1B) in the rat which exhibits high similarity with the ATIA receptor relative to amino acid sequence (95 % identity), binding of angiotensin II analogs, and utilization of Ca +2 as its intracellular second messenger. The adrenal and pituitary gland express primarily AT m mRNA whereas vascular smooth muscle and lung express primarily AT~A mRNA. Estrogen treatment suppressed ATIB but not ATxA mRNA levels in the pituitary gland. Thus, the unexpected existence of two putative ATx receptor genes appears to be related to the differential regulation of their expression rather than to different functional properties of the encoded receptor proteins. ®1992 Academic Press, Inc.

Angiotensin II is an octapeptide derived from the renin-angiotensinogen system and evokes a broad spectrum of effects related to regulation of blood pressure and salt and water metabolism (1). These effects include stimulation of vasoconstriction, aldosterone secretion, eatecholamine release, drinking, prolactin and ACTH secretion, and glycogenolysis (1). Angiotensin II activates specific receptors on artefiolar smooth muscle, adrenal cortex, adrenal medulla, brain, anterior pituitary, and liver, respectively, to achieve these effects (2).

The

primary signal transduction system associated with angiotensin II interaction with its receptor is increased phosphoinositide turnover with subsequent release of intracellular Ca2+ (2). Angiotensin II receptors are classified as type 1 (AT 0 or type 2 (AT2) based on their differential affinity for two non-peptide antagonists (Dup 753 and PD 123319, respectively) (3,4). AT~ receptors mediate the classical functions assigned to angiotensin II described above whereas the functions of AT 2 receptors remain unassigned (3,4). A rat AT~ receptor cDNA was cloned recently and shown to be a member of the 7transmembrane, G-protein coupled family of receptors (5,6). 0006-291X/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Here, we report the cloning,

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sequencing, and expression of a previously unsuspected second type of AT 1 receptor cDNA from the rat pituitary gland which appears to be encoded by its own gene.

To comply with

recommendations concerning the nomenclature of angiotensin II receptor subtypes (4), we use AT1B in reference to our receptor sub-type and ATIA in reference to the receptor sub-type described by Murphy et al. (5) and by Iwai et al. (6). Material and Methods

Preparation and screening of eDNA library: Poly(A) + RNA was prepared from the anterior pituitary glands of adult female rats. cDNA was prepared using oligo(dT) primers with the Stratagene kit. cDNAs ( > 0.5 kb) were ligated directionally into the x ZAP II vector such that transcription using T 3 RNA polymerase would yield sense RNA. A library of 8.5 x 106 independent clones was constructed ( > 95 % recombinant) and divided and amplified individually in 50 sub-libraries. RNA transcription for subsequent injection into Xenopus oocytes (7,8) was performed using the kit from Stratagene. Two days later the oocytes were voltage-clamped (7,8) in the presence of 5 ~M gonadotropin-releasing hormone. Positive pools of clones were 5-fold serially divided and tested (7,8) until a single clone was isolated that produced positive responses to GnRH in all oocytes. The Bluescript plasmid was then rescued from the ~ phage by in vivo excision and single-stranded cDNA prepared using helper phage R-408 (Stratagene). Nucleotide sequencing was performed on both strands using the Sequenase 2.0 kit (U.S. Biochemical) and synthetic oliognucleotide primers. The sequence analyses presented were performed using the Wisconsin GCG program on a VAX computer. Binding Assay:

The 2.1 kb AT m cDNA was subcloned directionally into the eukaryotic expression vector, pcDNA I (Invitrogen). COS-7 cells grown on 60 mm Petri dishes were transfected with 10 /~g of CsC1 purified plasmid DNA using 30 /~1 of lipofectin (Bethesda Research Labs) for 24 hr. After 48-60 hr, the cells were washed with PBS, scraped off the dishes, and homogenized in buffer (50 mM Tris-HC1, pH 7.4) to prepare cell membranes for angiotensin II receptor radioassay as described by Hauger et al. (9). Nonspecific binding, determined in the presence of 10-6M unlabelled angiotensin II, was approximately 10% of the total binding.

Calcium Measurements: COS-7 cells were transfected with the ATl~ cDNA-bearing plasmid, collected -65 hr later, and attached to glass cover slips coated with poly-L-lysine. The cells were incubated at 22°C in 4 ~M Fura-2/AM for 60 min (10). Fura-2 fluorescence ratios (F340/F38o) were collected from measurement windows placed around the circumference of each of 12 cells within the field of a video camera attached to a Zeiss IM-35 inverted microscope, as described in detail (10). Reverse Transcriptase/PCR: Poly(A +) RNA (1 /~g) from various tissues and cultured aortic vascular smooth muscle cells and total RNA (2 tzg) from anterior pituitary glands were subjected to first strand cDNA synthesis in a 40 /~1 reaction volume using oligo(dT) primer and AMV reverse transcriptase (Promega) for 60 rain. at 42°C. Various amounts (1,2,5 or 10/~1) of this solution were then used in a polymerase chain reaction (PCR) using the GeneAmp kit (Cetus) to produce a dose-product curve; the results presented are derived from the 5 ~1 samples since they were all within the linear range of the curve. The primers used for ATIB cDNA are shown in Fig. 2a as overlines 1 (sense) and 2 (antisense) and for ATIA cDNA as underlines 3 (sense) and 4 (antisense). The reaction conditions for PCR were 2 rain. at 96°C, 1.5 rain. at 64°C, and 4 rain. at 72°C for 28 cycles in a Perkin-Elmer-Cetus DNA thermal cycler. Ten/~1 of the 100 ~zl reaction mixture then was electrophoresed through a 1.0% agarose gel and stained with ethidium bromide. The identity of the DNA generated by PCR was confirmed by Southern blot analysis using a labelled probe representing the open reading frame of AT1B cDNA. 1091

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Animals and hormone treatment: Two independent groups of rats (5-8 rats/treatment in each

group) were surgically ovariectomized and then used after 2 weeks (data presented in this Figure) or 4 weeks (data not presented). Rats received two subcutaneous injections at 24 hr. intervals of sesame oil (OVX), 25 #g of 17-/3 estradiol benzoate in oil (OVX + E2) or 12.5 mg of progesterone in oil (OVX + P); anterior pituitary glands were collected 24 hr. after the second injection. Results and Discussion

Our cloning of the AT1B receptor cDNA occurred serendipitously while trying to isolate the receptor for gonadotropin releasing hormone (GnRH). A xZAP II cDNA library prepared from rat anterior pituitary RNA was screened in Xenopus oocytes using the standard two electrode voltage clamp procedure (7,8). Thirty two oocytes injected with 150 ng RNA derived from the isolated cDNA clone showed a mean 94 nA response to 5 x 10-6M GnRH while a similar number of uninjected oocytes showed no responses ( < lnA). However, when the cDNA was transfected into COS-7 cells, no binding of ~25I-D-Lys6-GnRH could be demonstrated. Nucleotide analysis of the cDNA insert revealed a sequence (Fig. 1) that was highly similar with the rat AT~A receptor sequences recently reported (5,6). Radioreceptor assays for angiotensin II on our transfected COS-7 cells confirmed that we, indeed, had cloned an AT m receptor rather than the GnRH-receptor (Fig. 2). We are unsure of the mechanism by which ATIB RNA induces GnRH-responsiveness in .Xenopus oocytes.

It may potentiate the signalling pathways of otherwise undetectable

endogenous GnRH-receptors since that is the mechanism by which RNA derived from members of the mas oncogene family are reported to induce angiotensin II responsiveness in Xenopus oocytes (11). Whatever the explanation, our results and those obtained with the mas oncogene (11) serve as warning to others using this popular expression cloning system that the RNA injected may not encode the activity measured in Xenopus oocytes. The AT1B receptor cDNA is composed of 2153 nucleotides [exclusive of the poly(A+) tail] and exhibits 74 % identity with the AT~A receptor cDNA overall and 91% identity with the open reading frame (Fig. la). The open reading frames of both the AT1B and AT1A cDNAs encode proteins comprised of 359 amino acids which exhibit a 95% level of identity (Fig. lb). To confirm the nucleotide sequence of AT1B cDNA, we isolated 7 additional independent clones from the pituitary cDNA library using a labelled cDNA probe representing the open reading frame of ATIB; 250-300 nucleotides were sequenced beginning at the 3" end (7 clones) and the 5" end (4 clones). Six of the 3" sequences were identified as AT1B and one as AT1A. All four of the 5" sequences were ATIB and each originated at the 5 "nucleotide shown in Fig. la; this finding suggests that we have correctly identified the 5" terminus of the AT m cDNA and that the ATIA cDNA has an additional 141 nucleotides at its 5" terminus.

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Vol. 183, No. 3 , 1 9 9 2 a

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FIG. l. Comparison of the nucleotide and derived amino acid sequences of angiotensin II receptors AT~B and AT]a. a, Nucleotide sequences that are identical in the AT m and AT]~ cDNAs are shaded; dots are gaps introduced to optimize alignment of the sequences. Nucleotides comprising the open reading frame are capitalized. The over- and under-lines 1-4 are the sequences of synthetic olignucleotides used for the polymerase chain reactions described in Fig. 4. b, Derived amino acid sequences of the AT m and AT]~ receptors. Dashes in the AT~a sequence indicate identity with the AT]B receptor. The overlines I-VII represent predicted transmembrane regions. Asparagine-linked glycosylation sites are marked with an asterisk; predicted extracellular cysteines are indicated by inverted triangles; and potential sites for phosphorylation by protein kinase C are marked with closed dots. 1093

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100

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FIG. 2. Unlabelled ligand displacement curves in an 125I-angiotensinII radioreceptor assay on membranes prepared from COS-? cells transfected with ATIa receptor cDNA. The results shown are from one of 3 radioreceptor assays performed on each of 3 independent transfections; each point is the result of triplicate determinations.

The pattern of nucleotide and amino acid differences between the ATIB and ATIA receptor cDNAs is not characteristic of alternatively spliced RNA, suggesting that they are encoded by different genes.

Indeed, at least two fragments of rat genomic DNA treated with multiple

restriction enzymes hybridized under high stringency conditions with an AT1B cDNA probe representing the open reading frame (data not shown). Angiotensin II receptor radioassays performed on COS-7 cells transfected with the AT1B cDNA revealed a pattern of ligand displacement similar with that of the AT1A receptor (Fig. 2) except for angiotensin I which is approximately 10-fold less potent than with the ATaA receptor (5,6). Our receptor clearly is an AT 1 type since Dup 753, a non-peptide antagonist specific for type 1 receptors (3,4), has an IC50 value of ~3x10-9M whereas PD 123319, a nonpeptide antagonist specific for type 2 receptors (3,4), is inactive at lxl0-6M. Scatchard analysis revealed a Kd (0.7 nM) similar with those observed for the ATIA receptor (0.68 -1.9 nM) (5,6) and for the endogenous anterior pituitary angiotensin II receptor (0.95 nM) (12). AT 1 receptors in cells from various tissues (13) including the anterior pituitary gland (14) are reported to be functionally coupled to increases in intracellular Ca ÷2. Thus, we used COS-7 cells transfected with the AT~B cDNA for measurements of Ca +2 levels using video-based fluorescence ratio imaging of the calcium indicator dye, Fura-2 (10).

About 14% of the

transfected COS-7 cells responded to angiotensin II; they exhibited a mean 2.2-fold peak increase in the Fura-2 fluorescence ratio (Fig. 3). None of the cells transfected with the control pcDNA-I plasmid responded to angiotensin II. The AT1A receptor also was reported to be functionally coupled to increases in intracellular calcium (5,6). 1094

Vol. 183, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 0.60 0.50

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FIG. 3. Angiotensin II-stimulated increases in intracellular Ca +2 levels in COS-7 cells transfected with the ATIB receptor. In 3 independent transfections with AT1BcDNA, 23 of 168 COS-7 cells (13.7%) responded to angiotensin II (10-7M); none of 48 cells responded when transfected with the control pcDNA I plasmid.

The high level of identity of the two AT x cDNAs resulted in cross-hybridization of ATIB cDNA with AT1A mRNA and vice-versa. Tissue measurements of AT1B and AT1A mRNA levels, therefore, were performed using reverse transcriptase/polymerase chain reactions (15) with short, dissimilar oligonucleotide primers derived from the 5" and 3" untranslated regions of their cDNAs (Fig. la). Anterior pituitary, adrenal, and uterus express primarily AT1B mRNA whereas aortic vascular smooth muscle cells, lung, and ovary express primarily AT1A mRNA; spleen, liver, and kidney express similar levels of the two RNAs (Fig. 4a).

a

Pit

Ad

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ATIA

FIG. 4. AT1Band ATIA mRNA expression in various tissues (a) and their hormonal regulation in the anterior pituitary gland (b). Ethidium bromide stained DNA is shown which was generated from tissue mRNA using reverse transcriptase/polymerase chain reactions. In a: Pit, anterior pituitary gland; Ad, adrenal gland; VSMC, vascular smooth muscle cells; Sp, spleen; Lv, liver; Lu, lung; Kd, kidney, Ut, uterus; Ov, ovary. In b: CONTROL, intact rats; OVX, ovariectomized rats; OVX + E2, estrogen-treated ovariectomized rats; OVX + P, progesteronetreated ovariectomized rats. 1095

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Angiotensin II receptor levels in the anterior pituitary gland are reported to be strongly inhibited (75-90%) by estrogens (12). In Fig. 4b we show that estrogen exerts a similar effect on AT m but not ATIAmRNA levels in this tissue. Ovariectomy resulted after two weeks in no increase in AT m mRNA levels (Fig. 4b) whereas after four weeks a large increase was observed (data not shown). Estrogen, but not progesterone treatment after ovariectomy, resulted in large decreases in AT m mRNA levels in both groups of ovariectomized animals (2 weeks, Fig. 4b; 4 weeks, data not shown). The existence of two AT~ receptor subtypes was not previously suspected probably due to the high similarity of function of the encoded proteins. Indeed, their high level of amino acid identity (95 %) appears to be unprecedented among the large family of 7-transmembrane receptors (16,17). Differences in mRNA tissue distributions and in estrogen-regulation suggest that the existence of AT1A and AT m genes is related to regulation of their expression rather than to different functional properties of the encoded proteins. This same principle may apply to the plethora of receptor subtypes which has become a characteristic feature of most of the 7transmembrane, G-protein receptors described (16,17). References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Peach, M.J. (1977) Physiol. Rev. 57, 313-370. Vallotton, M. B. (1987) Trends Pharmacol. Sci. 8, 69-74. Chiu, A. T., Herblin, W. F., McCall, D.F., Ardecky, R.J., Carini, D.J., Duncia, J.V., Pease, L. J., Wong, P. C., Wexler, R. R., Johnson, A. L., and Timmermans, P.B.M. W. M. (1989) Biochem. Biophys. Res. Commun. 165, 196-203. Bumpus, F.M., Catt, K. J., Chiu, A. T., De Gasparo, M., Goodfriend, T., Husain, A., Peach, M.J., Taylor, Jr., D. G., and Timmermans, P. B. M. W. M. (1991) Hypertension 17, 720-721. Murphy, T. J., Alexander, R. W., Griendling, K. K., Runge, M.S. & Bernstein, K.E. (1991) Nature 351,233-236. Iwai, N., Yamano, Y., Chaki, S., Konishi, F., Bardhan, S., Tibbetts, C., Sasaki, K., Hasegawa, M., Matsuda, Y., and Inagami, T. (1991) Biochem. Biophys. Res. Commun. 177, 299-304. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., and Nakashini, S. (1987) Nature 329, 836-838. Kushner, L., Lerma, J., Bennett, M. V. L., and Jukin, R. S. (1989) In Methods in Neurosciences (Conn, P. M. Ed.) pp. 3-29. Academic Press, San Diego. Hauger, R. L., Aguilera, G., Baukal, A. J., and Catt, K. J. (1982) Molec. Cell. Endocrinol. 25, 203-212. Morris, A. P., Kirk, K. L., and FrizzeU, R. A. (1990) Cell Regul. 1, 951-963. Monnot, C., Weber, V., Stinnakre, J., Bihoreau, C., Teutsch, B., Corvol, P., and Clauser E. (1991) Molec. Endocrinol. 5, 1477-1487. Chen, F-C. M., and Printz, M. P. (1983) Endocrinology 113, 1503-1510. Peach, M. J., and Dostal, D. E. J. (1990) Pharmacol. Sci. 16 (Suppl. 4), 525-530. Jones, T. H., Brown, B. L., and Dobson, P. R. M. (1988) J. Endocrinol. 116, 367-371. Wang, A. M,, Doyle, M. V., and Mark, D. F. (1989) Proc. Natl. Acad. Sci. 86, 97179721. Dohlman, H. D., Thomer, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653-688. Attwood, T. K., Eliopoulos, E. E., and Findlay, J. B. C. (1991) Gene 98, 153-159. 1096