Cloning and expression of the mouse homolog of the human alpha 2-C2 adrenergic receptor.

Three subtypes of alpha 2 adrenergic receptors have been identified in the human and rat. The subtype located on human chromosome 2 (alpha 2-C2) is unique in that it is expressed mainly in the peripheral tissues and lacks sites for N-linked glycosylation. We isolated the gene encoding the mouse homolog of the human alpha 2-C2 adrenergic receptor (M alpha 2-2H). The deduced amino acid sequence of the M alpha 2-2H shows 82% and 96% identity to the human alpha 2-C2 and the rat RNG alpha 2 adrenergic receptors, respectively. Southern blot analysis demonstrated that the M alpha 2-2H was encoded by a single copy gene and was distinct from the mouse homologs of the alpha 2-C4 and alpha 2-C10 adrenergic receptors. When expressed in COS-7 cells, the M alpha 2-2H exhibited a pharmacological profile similar to the human alpha 2-C2 and rat RNG alpha 2 receptors.     

Functional interactions of recombinant alpha 2 adrenergic receptor subtypes and G proteins in reconstituted phospholipid vesicles.

The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.     

A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice

We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse pro alpha 2 (I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse pro alpha 2 (I) collagen gene, cloned upstream of either the Escherichia coli beta- galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal pro alpha 2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse pro alpha 1(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro alpha 2(I) collagen gene.     

Physical linkage of mouse lambda genes by pulsed-field gel electrophoresis suggests that the rearrangement process favors proximate target sequences.

The first complete map of a mammalian immunoglobulin gene locus is presented. Mouse lambda genes were mapped by pulsed-field gel electrophoresis. The gene order is V2-Vx-C2-C4-V1-C3-C1. The distance between V2 or Vx and the C2-C4 cluster is 74 or 55 kilobases (kb), respectively, whereas that between V1 and C3-C1 is only 19 kb; V2 and C3-C1 are at least 190 kb apart. Thus, the distances between the lambda subloci are inversely proportional to their frequencies of rearrangement. The related gene lambda 5 is not within the 500 kb of the lambda locus mapped here.     

Stable expression of recombinant human alpha 2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay.

Cloning of the genes encoding distinct subtypes of human alpha 2-adrenergic receptors (alpha 2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human alpha 2-AR subtypes alpha 2-C4 and alpha 2-C10 at densities of approx. 2 x 10(5) receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5'-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the alpha 2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding.     

Structural analysis of a rabbit immunoglobulin kappa 2 J-C locus reveals multiple deletions.

We previously reported that domestic rabbits harbor at least two DNA sequences that hybridize strongly to immunoglobulin kappa C region probes in Southern blots. One of these was cloned from a domestic b4 rabbit and identified as the gene for the nominal b4 allotype kappa chain which is expressed at high levels. We now have cloned (from a b4 rabbit) the other homologous sequence and find that it encodes a kappa chain nearly identical to the kappa 2 chain of "bas" rabbits, which is not normally expressed at detectable levels in domestic rabbits. Sequence analysis of this kappa 2 chain reveals a J kappa -C kappa locus with no obvious coding sequence defects that could explain its low expression. However, several base changes in a putative enhancer region as well as deletions (totalling about 1.5 kb) in the J-C intron might be related to low expression. The comparison between these two kappa genes raises questions about the selective pressures operating during the evolution of this gene system.     

Isolation of rat genomic clones encoding subtypes of the alpha 2-adrenergic receptor. Identification of a unique receptor subtype.

alpha 2-Adrenergic receptors (alpha 2-AR) exist as subtypes that are expressed in a tissue-specific manner and differ in 1) their ligand recognition properties, 2) their extent of receptor protein glycosylation, and possible 3) their mechanism of signal transduction. Genomic or cDNA clones encoding three receptor subtypes have been characterized; however, both functional and radioligand binding studies in rodents suggest the existence of a fourth receptor subtype. To isolate the rat genes encoding receptor subtypes we screened a rat genomic library with an oligonucleotide probe encompassing the third membrane span of the human C-4 alpha 2-AR. Two intronless rat genes were isolated that encode distinct receptor subtypes (RG10, RG20). RG10 and RG20 encode proteins of 458 and 450 amino acids, respectively, that are 56% homologous and possess the structural features expected of this class of membrane-bound receptors. RG10 identifies a mRNA species of approximately 2500 nucleotides that is found primarily in brain, whereas RG20 identifies a larger mRNA species (approximately 4000 nucleotides) that is found in several tissues including brain, kidney, and salivary gland. RG10 is 88% homologous to the human C-4 alpha 2-AR and exhibits similar binding properties ( [3H]rauwolscine KD = 0.7 +/- 0.3 nM) as determined following transient expression of the receptor in COS-1 cells. RG20 exhibits ligand binding properties distinct from the three receptor subtypes identified by molecular cloning. Saturation binding studies indicate an affinity constant of 15 +/- 1.2 nM for the alpha 2-AR antagonist [3H]rauwolscine, a value 6-20 times higher than that observed for the three cloned receptor subtypes. In competition binding studies the potency order of competing ligands for RG20 is phentolamine greater than idazoxan greater than yohimbine greater than rauwolscine greater than prazosin. Of the three previously cloned alpha 2-AR, RG20 is most closely related to the human C-10 alpha 2-AR (89% homology) and is also capable of mediating adenylylcyclase inhibition as determined following its stable expression in NIH-3T3 fibroblasts. However, in contrast to RG20, [3H] rauwolscine exhibits a KD of 2 nM for the C-10 receptor, and the potency order for competing ligands is rauwolscine greater than or equal to yohimbine greater than idazoxan greater than phentolamine greater than prazosin. RG20 and C-10 are also distinguished by their affinity for SKF-10478 (RG20 Ki = 531 nM, C-10 Ki = 101 nM), a compound that may functionally distinguish pre- and postsynaptic alpha 2-AR. These data suggest that RG20 represents a fourth alpha 2-AR subtype distinct from the known alpha 2A-C receptor subtypes.     

High level of expression of functional human platelet alpha 2-adrenergic receptors in a stable mouse C127 cell line

We have generated, by transfection and proper selection, a stable mouse C127 cell line which expresses the human alpha 2-adrenergic receptor gene. The size of the mRNA produced by the cloned gene is 1.8 kb. Electrophoretic analysis and autoradiography of cell membrane proteins photoaffinity labeled with p-[3H]azidoclonidine gave a broad protein band of molecular mass of approx. 64 kDa. Saturation binding with [3H]rauwolscine as ligand gave an equilibrium dissociation constant of 1.29 +/- 0.46 nM (mean +/- S.D.) and binding capacity range of 18-35 pmol/mg membrane protein, with (3-6) x 10(6) receptors per cell. Antagonist competition experiments displayed the order of potency: yohimbine greater than rauwolscine greater than phentolamine much greater than prazosin. Agonist competitions demonstrated the order of potency: p-aminoclonidine greater than (-)epinephrine much greater than (+)epinephrine much greater than (-)isoproterenol. This pharmacological profile is characteristic of the human platelet alpha 2-adrenergic receptor. The expressed receptor is able to couple to the Gi protein. Thus, when epinephrine competition for specific binding of [3H]rauwolscine was performed in the presence of 1 mM MgCl2, 1 mM Gpp[NH]p increased the Ki for epinephrine from 164 to 315 nM. Following preincubation of cultures with 1 mM isobutylmethylxanthine, 1 microM epinephrine decreased forskolin-stimulated cellular cyclic AMP accumulation by 72%. The response was biphasic, and the attenuation effect disappeared at 100 microM epinephrine. A transfected clone which did not demonstrate detectable alpha 2-adrenergic receptor mRNA displayed low levels of alpha 2-adrenergic receptor, (less than 50 fmol/mg membrane protein), similar to those found in the parent C127 cell line. In this clone, epinephrine did not attenuate but, rather, enhanced forskolin-stimulated cyclic AMP accumulation. This new C127 cell line expressing high levels of alpha 2-adrenergic receptor provides an abundant source of a single human adrenergic receptor subtype in membrane-bound conformation which is able to couple to the Gi protein and inhibit forskolin-stimulated adenylate cyclase activity. This cell line will facilitate studies of the structure: function relationship of the alpha 2-adrenergic receptor and should aid in separating the components of various signal transduction mechanisms putatively attributed to this receptor.     

Four synonymous genes encode calmodulin in the teleost fish, medaka (Oryzias latipes): conservation of the multigene one-protein principle.

We cloned four distinct calmodulin (CaM)-encoding cDNAs from a small teleost fish, medaka (Oryzias latipes). The deduced amino acid (aa) sequences were exactly the same in these four genes and identical to the aa sequence of mammalian CaM, because of synonymous codon usages. The four cDNAs from medaka, termed CaM-A, -B, -C and -D, corresponded to mRNAs of 1.8, 1.4, 2.5 and 1.8 kb, respectively, in Northern blot analysis. Our results demonstrated that the 'multigene one-protein' principle of CaM synthesis is applicable to medaka, as well as to mammals whose CaM is encoded by at least three different genes.