Abstract
The rat cA2-47 gene encodes the pharmacologically defined α2D-adrenergic receptor (α2D-AR) subtype. Previously, the expression of its mRNA was shown in bovine retina by amplification through the reverse transcription-polymerase chain reaction (RT-PCR) of a region corresponding to the rat α2D-AR, amino acid (aa) residues 382–439, indicating the presence of this subtype in this neural tissue. In the present study, the structure of this gene has been probed and the encoded receptor subtype has been characterized in bovine retina and its photoreceptor cells. The deduced as sequence of the two bovine gene fragments, aa residues 290–375 and as residues 392–434, demonstrates 77% overall identity with the rat α2D AR subtype and 80% overall identity with the mouse α2D-AR. The receptor encoded by the bovine gene was expressed in the retina and its photoreceptors with the typical pharmacological characteristics established for the rat (α2D-AR subtype: The receptor bound rauwolscine with a KD of 14 nM in the retina and with that of 19 nM in the photoreceptor cells; the binding association rate constant, k+1, for the ligand was 0.012 min−1, the dissociation rate constant, k−1, was 0.14 min−1 and the half-time for dissociation was 5 min. Oxymetazoline displaced the bound [3H]-rauwolscine with an EC50 value of 85 nM, while SK & F 104078, and prazosin displaced the bound [3H]-rauwolscine with the respective IC50 values of 900 nM and 3000 nM. The other α2-AR subtypes − α2B-AR, α2C-AR — were not detected in the retina and its photoreceptors. Thus, this study shows that the bovine α2D AR gene is a structural variant of the rat and mouse genes, that the bovine gene encodes the typical pharmacologically defined α2D-AR. subtype, that this subtype is present in its exclusive form in the bovine retina and its photoreceptors, where it may be presynaptic in nature.
Similar content being viewed by others
References
Kobilka BK, Matsui H, Kobilka TS, Yang-Feng TL, Francke U, Caron MG, Lefkowitz RJ, Regan JW: Cloning, sequencing, and expression of the gene coding for the human platelet α2-adrenergic receptor. Science 238, 650–656, 1987
Regan JW, Kobilka TS, Yang-Feng TL, Caron MG, Lefkowitz RJ, Kobilka BK: Cloning and expression of a human kidney cDNA for an α2-adrenergic receptor subtype. Proc Natl Acad Sci USA 85: 6301–6305, 1988
Lomasney JW, Lorenz W, Allen LF, King K, Regan JW, Yang-Feng TL, Caron MG, Lefkowitz RJ: Expansion of the α2-adrenergic receptor subtype, the gene for which is located on chromosome 2. Proc Natl Acad Sci USA 87: 5094–5098, 1990
Weinshank RL, Zgombic JM, Machhi M, Adham N, Lichtblau T, Branchek TA, Hartig PR: Cloning, expression and pharmacological characterization of a human α2B-adrenergic receptor. Mol Pharmacol 38:681–688, 1990
Kurose H, Arriza JL, Lefkowitz RJ: Characterization of a2adrenergic receptor subtype-specific antibodies. Mol Pharmacol 43: 444–150, 1993
Bylund DB, Ray-Prenger C, Murphy TJ: Alpha-2A and alpha-2B adrenergic receptor subtypes: antagonist binding in tissues and cell lines containing only one subtype. J Pharmacol Exp Ther 245: 600–607, 1988
Blaxall HS, Murphy TJ, Baker JC, Ray C, Bylund DB: Characterization of the alpha-2C adrenergic receptor subtype in the opossum kidney and in the OK cells. J Pharmacol Exp Ther 259: 323–339, 1991
Simmoneaux V, Ebadi M, Bylund DB: Identification and characterization of ago adrenergic receptors in bovine pineal gland. Mol Pharmacol 40: 235–241, 1991
Michel AD, Loury DN, Whiting RL: Differences between α2-adrenoreceptor in rat submaxillary gland and the α2A- and α2B adrenoreceptor subtypes. Br J Pharmacol 98: 890–897, 1989
Chalberg SC, Duda T, Rhine JA, Sharma RK: Molecular cloning, sequencing, and expression of an α2-adrenergic receptor complementary DNA from rat brain. Mol Cell Biochem 97: 161–172, 1990
Lanier SM, Downing S, Duzic E, Homey CJ: Isolation of rat genomic clones encoding subtypes of the α2-adrenergic receptor. Identification of a unique receptor subtype. J Biol Chem 266: 10470–10478, 1991
Wypijewski K, Duda T, Sharma RK: Structural, genetic and pharmacological identity of the rat α2-adrenergic receptor subtype cA2-47 and its molecular characterization in rat adrenal, adrenocortical carcinoma and bovine retina. Mol Cell Biochem 144: 181–190, 1995
Berlie JR, Iversen LJ, Blaxall HS, Cooley ME, Chacko DM, Bylund DB: Alpha-2 adrenergic receptors in the bovine retina. Presence of only the alpha-2D subtype. Invest Opthalmol Vis Sci 36: 1885–1892, 1995
Chomczynski P, Sacchi N: Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 151–159, 1987
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. John Wiley & Sons Inc. USA., 1987, p 491–196
Sitaramayya A: Rhodopsin kinase prepared from bovine rod disk membranes quenches light activation of cGMP phosphodiesterase in a reconstituted system. Biochemistry 25, 5460–5468, 1986
Ren Q, Kurose H, Lefkowitz RJ, Cotecchia S: Constitutively active mutants of the α2-adrenergic receptor. J Biol Chem 268: 16483–16487, 1994
Dalman HM, Neubig RR: Two peptides from the α2A-A-adrenergic receptor after receptor G protein coupling by distinct mechanisms. J Biol Chem 266: 11025–11029, 1991
O'Rourke MF, Iversen LJ, Lomasney JW, Bylund DL: Species orthologs of the alpha-2A adrenergic receptor: The pharmacological properties of the bovine and rat receptors differ from the human and porcine receptors. J Pharm Exp Ther 271: 735–740, 1994
Akers I, Coates J, Drew GM, Sullivan AT: α2-Adrenoreceptor blocking profile of SK & F 104078: further evidence for receptor subtypes. Br J Pharmacol 102: 943–949, 1991
Hieble JP, Sulpkio AC, Nichols AJ, Willette, Ruffolo RR: Pharmacological characterization of SK & F 104078, a novel alpha-2 adrenoreceptor antagonist that discriminates between pre- and postjunctional alpha-2 adrenoreceptors. J Pharmacol Exp Ther 247: 654–652, 1988
Daly RN, Ashton D, Sulpkio AC, Levitt B, Demarinas RM, Regan JW, Ruffolo RR, Hieble JP: Evidence for heterogeneity between preand postjunctional α2-adrenoreceptors using 9-substituted 9-benzazapines. J Pharmacol Exp Ther 247, 122–128, 1988
Link R, Daunt D, Barsh G, Chruscinski A, Kobilka BK: Cloning of two mouse gene encoding α2-adrenergic receptor subtypes and identification of a single amino acid in the mouse α2-C 10 homolog respon sible for an interspecies variation in antagonist binding. Mol Pharmacol 42: 16–27, 1992
Fraser CM, Arakawa S, McCrombie W, Venter JC: Cloning, sequence analysis, and permanent expression of a human alpha2-adrenergic receptor in chinese hamster ovary cells. Evidence for independent path ways of receptor coupling to adenylate cyclase attenuation and activation. J Biol Chem 264: 11754–11761, 1989
Guyer CA, Horstman DA, Wilson AL, Clark JD, Cragoe Jr EJ, and Limbird LE: Cloning, sequencing and expression of the gene encoding the porcine α2-adrenergic receptor. J Biol Chem 265: 17307–17317, 1990
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Venkataraman, V., Duda, T., Galoian, K. et al. Molecular and pharmacological identity of the α2D-adrenergic receptor subtype in bovine retina and its photoreceptors. Molecular and Cellular Biochemistry 159, 129–138 (1996). https://doi.org/10.1007/BF00420915
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00420915