Molecular cloning, sequencing and expression of an ?2-adrenergic receptor complementary DNA from rat brain

Abstract

We have isolated a cDNA clone from rat brain using a human platelet alpha 2-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney alpha 2-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3'-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5'-untranslated region. Southern-blot analysis with a human platelet alpha 2-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain alpha 2-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A+) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the alpha 2-adrenergic antagonist [3H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the alpha 2A subclass. We conclude that the rat brain cDNA encodes a new alpha 2-adrenergic receptor subtype that may be brain-specific.

Assessment of the role of α2‐adrenoceptor subtypes in the antinociceptive, sedative and hypothermic action of dexmedetomidine in transgenic mice

“…These correspond to the cloned a 2 -C10, a 2 -C2 and a 2 -C4 adrenoceptors, designated according to human chromosomal localization (Kobilka et al, 1987;Regan et al, 1988;Weinshank et al, 1990) with orthologues (RG20, RNG and RG10) in the rat (Chalberg et al, 1990;Zeng et al, 1990;Lanier et al, 1991). The a 2D AR subtype was determined to be a rat homologue of the human a 2A AR.…”

Delineation of three pharmacological subtypes of α2‐adrenoceptor in the rat kidney

“…The relations of our a2-adrenoceptors to those for which the gene structures have been determined (Kobilka et al, 1987a;Regan et al, 1988;Lomasney et al, 1990;Chalberg et al, 1990;Zeng et al, 1990;Voigt et al, 1991) are not entirely clear. Our two a2B-adrenoceptors show similar pharmacology to the expressed rat a2-RNG and rat a2-pA2d receptors (Zeng et al, 1990;Voigt et al, 1991) due to their similar high affinities for yohimbine and prazosin and low affinities for oxymetazoline, but available data does not allow definite classification of either a2B1 or a2B2 as being identical to the a2-RNG or CX2-pA2d receptors.…”

“…Very limited binding data are available for the a2-adrenoceptor cloned from rat brain (cA2-47 clone (Chalberg et al, 1990); 95, 71 and 71% homologous to a2-C10, x2-C4 and a2-C2, respectively) which makes it difficult to link this receptor with any of our present ones. Moreover, x2-cA2-47 and x2-C1O probes hybridized to different restriction fragments in the rat genome (Chalberg et al, 1990) possibly indicating that a2-cA2-47 may represent yet another a2-receptor subtype. Guanoxabenz, and ARC 239, in conjunction with [3H]-RX821002 binding and use of expressed receptor proteins may prove useful in linking structure to pharmacology for X2-adrenoceptors.…”

“…Subsequent studies then indicated that these receptors consisted of at least four classes for which the names at and a2 (see Berthelsen & Pettinger, 1977;Wikberg, 1978) and fil and P2 (Lands et al, 1967) were used. More recently the molecular cloning of the genes for adrenoceptors have indicated that there exist at least two a,-, three a2-and three distinct fiadrenoceptor genes in mammals which code for receptors with differing pharmacological properties (Dixon et al, 1986;Firelle et al, 1987;Kobilka et al, 1987a,b;Cotecchia et al, 1988;Regan et al, 1988;Emorine et al, 1989;Schwinn et al, 1990;Lomasney et al, 1990;Chalberg et al, 1990;Zeng et al, 1990;Voigt et al, 1991). However, although radioligand binding techniques and other functional methods have indicated the presence of multiple receptors of the x2-type (Maura et al, 1985;Bylund, 1985;1988;Adams et al, 1986;Ruffolo et al, 1987;Michel et al, 1989a;Uhlen et al, 1990) it has not yet been possible to merge the molecular biological data with the functional into a unifying hypothesis as to the number of distinct CZ2-adrenoceptor subtypes .…”

Testosterone Dependence of Salt-Induced Hypertension in Sabra Rats and Role of Renal α2-Adrenoceptor Subtypes

“…One cycle of PCR consisted of 1 min at 94°C, 1 min at 57°C, and 1 min at 72°C for ␣ 2 -adrenoceptor subtype cDNA. Primers used for amplification were as follows: ␣ 2A -, 5Ј primer: 5Ј-GCGCCCCA-GAACCTCTTCCT-3Ј and 3Ј primer: 5Ј-AGTGGCGGGAAGGAGAT-GAC-3Ј (Chalberg et al, 1990); and ␣ 2B -, 5Ј primer: 5Ј-AGCATCG-GATCTTTTTTTGC-3Ј and 3Ј primer: 5Ј-GTTTGGGGTTCACATT-CTTC-3Ј (Le Jossec et al, 1995). For ␤-actin, similar experimental conditions were used, except that the annealing temperature was 53°C.…”