Agonist interactions with chimeric and mutant beta1- and beta3-adrenergic receptors: involvement of the seventh transmembrane region in conferring subtype specificity.

beta1- and beta3-adrenergic receptors (AR) are the predominant beta-AR subtypes in adipocytes, and analysis of native and recombinant beta-AR has revealed several pharmacological and biochemical differences between these subtypes. This study used chimeric and mutated rat beta-AR expressed in Chinese hamster ovary cells to examine the basis of certain characteristic differences in the agonist properties of catecholamines and prototypic beta3-AR agonists. The exchange of sequence beyond transmembrane (TM) region 6 between the beta-AR subtypes had dramatic and reciprocal effects on the affinity and efficacy of the prototypic beta3-AR agonists BRL 37,344 and CL 316,243, without affecting the interactions with catecholamines. Mutation of Phe350 and Phe351 in TM7 of the beta1-AR to Ala and Leu found in the beta3-AR was sufficient to allow activation by prototypic beta3-AR agonists. Interestingly, this mutation did not affect catecholamine action and it did not impair the ability of propranolol to block the actions of isoproterenol or the selective beta3-AR agonists. beta1-AR containing beta3-AR sequence from predicted TM5 through TM6 exhibited reduced affinity for catecholamines without altering agonist potency, suggesting enhanced coupling efficiency. Inclusion of the homologous beta1-AR sequence in the beta3-AR, however, did not produce reciprocal effects. These results are the first to define a major determinant of beta3-AR subtype-selective agonism in TM7 and demonstrate that the determinants of selective phenethanolamines, catecholamines, and propranolol action are distinct.

Selective up-regulation of alpha1a-adrenergic receptor protein and mRNA in brown adipose tissue by neural and beta3-adrenergic stimulation.

Previous studies have shown that neural stimulation of brown adipose tissue (BAT) reorganizes the expression and activity of signaling proteins in the beta-adrenergic adenylyl cyclase pathway. Cold stress increases neural stimulation of BAT and increases alpha1-adrenergic receptor number; however, the alpha1 receptor subtype involved and the mechanism of up-regulation by cold stress have not been determined. Using reverse transcription/polymerase chain reaction analysis and nuclease protection assay, BAT was demonstrated to express mRNAs encoding alpha1a and alpha1d, but not alpha1b, receptors. Parallel pharmacologic studies of BAT membranes and recombinant alpha1a and alpha1d receptors expressed in COS-7 cells demonstrated that alpha1a receptors predominate in BAT. Exposure of rats to 4 degrees for 4 days increased alpha1a receptors and mRNA in BAT but did not alter expression of alpha1d receptors or mRNA. The induction of alpha1a receptor and mRNA level by cold stress was prevented by selective surgical denervation of BAT. Furthermore, alpha1a receptor and mRNA expression could be induced in warm-adapted rats by infusions of the selective beta3-adrenergic receptor agonist CL 316,243. These data indicate that neural activation of beta3-adrenergic receptors is an important determinant of alpha1a adrenergic receptor expression in BAT.

Regulation of mouse beta 3-adrenergic receptor gene expression and mRNA splice variants in adipocytes.

This study examined the regulation of murine beta 3-receptor mRNA and determined whether the recently described mRNA splice variants are differentially regulated by agents that alter total beta 3-receptor mRNA levels. In vivo treatment of mice with the beta 3-receptor agonist BRL-26830 reduced total beta 3-transcripts by 64% in white adipose tissue but did not alter the mRNA splicing pattern. Further analysis in cultured 3T3-F442A adipocytes showed that isoproterenol, dexamethasone, or phorbol 12-myristate 13-acetate also greatly reduced beta 3-receptor mRNA levels without selectively altering poly-U-containing transcripts. Blockade of transcription with actinomycin D produced a rapid loss of beta 3-receptor mRNA, which was prevented by blockade of mRNA translation with cycloheximide. However, neither actinomycin D nor cycloheximide altered the splicing pattern of beta 3-receptor mRNA. Analysis of transcription rate by nuclear run-off assay indicated that 8-bromoadenosine 3',5'-cyclic monophosphate and phorbol 12-myristate 13-acetate reduce beta 3-receptor gene transcription and that suppression of transcription is sufficient to account for the reduction in beta 3-receptor mRNA levels by these agents.

Analysis of human and rodent beta 3-adrenergic receptor messenger ribonucleic acids.

Recent evidence indicates that the human and rodent beta 3-receptor genes differ with respect to their intron/exon organization and putative regulatory elements in the proximal promotor regions. The present work examined the molecular basis for heterogeneity among human and rodent beta 3-receptor messenger RNAs (mRNAs) and mRNA transcription start site selection with gene-derived human and rodent probes. This analysis indicates that the mRNA size heterogeneity of the human beta 3-receptor mRNA seen in Northern blots results from the use of alternative polyadenylation signals. In contrast, mRNA heterogeneity of the rat beta 3-receptor mRNA is due to the use of widely separated promoters. Analysis of mRNA transcription sites within the proximal promoters of the human and rat genes indicates that multiple homologous start sites are used. However, the pattern of transcription initiation appears to be both species and tissue specific. In rat adipose tissues where abundant expression of the beta 3 gene occurs, most transcripts began at -161 relative to translation initiation ATG, whereas minor transcription began around -123 and -109. Interestingly, virtually all transcripts were found to begin at the -123 and -109 sites in the rat gastric fundus, which expresses only low levels of beta 3-receptor mRNA. In human brown fat and neuroepithelioma cells, the vast majority (> 80%) of beta 3 transcripts began at multiple sites around -130. Exposure of murine 3T3-F442A adipocytes to isoproterenol, 8-bromo-cAMP, phorbol 12-myristate 13-acetate, or dexamethasone dramatically reduced murine beta 3-receptor mRNA levels. In contrast to mouse adipocytes, 8-bromo-cAMP increased human beta 3-receptor mRNA levels in SK-N-MC neuroepithelioma cells, whereas isoproterenol, phorbol 12-myristate 13-acetate, and dexamethasone were without effect. These data identify important differences in the structure and regulation of the human and rodent beta 3-receptor genes and suggest that the data obtained in rodent models may not be directly applicable to the regulation of the human gene.

Characterization of the human beta 3-adrenergic receptor gene.

Comparison of the rodent and human beta 3-adrenergic receptor cDNAs with the respective genomic sequences has revealed unexpectedly that these genes contain two protein-coding exons. The rat gene was cloned recently and was found to contain three exons and two introns. In the present report, the human beta 3 receptor gene was characterized and was found to consist of two exons and a single intron. Sequence analysis of the human beta 3 receptor gene identified regions in the intron that were homologous to the second exon and second intron of the rat gene. It appears that both species utilize homologous 5' donor sites in the first intron and 3' acceptor sites of the final exon. However, splicing signals within the human intron that are homologous to the second exon of the rat gene are not used. Nuclease protection assays of tissue RNA and polymerase chain reaction-amplified cDNA demonstrated conclusively that beta 3 receptor mRNA, containing two protein-coding exons, is expressed in human adipose and intestinal tissues. The pharmacological properties of the full length human beta 3 receptor were determined for the first time in Chinese hamster ovary cells, where catecholamine agonists activated adenylyl cyclase with low potency. The beta 3 receptor agonists CGP 12177 and BRL 37344 also activated adenylyl cyclase. CGP 12177 was 10-15 times more potent than either isoproterenol or BRL 37344 in stimulating adenylyl cyclase activity. These pharmacological properties differed somewhat from those reported previously for Chinese hamster ovary cells expressing the truncated receptor. However, direct comparison indicates that it is unlikely that the amino acid sequence derived from the second exon can account for these differences.

Rodent and human beta 3-adrenergic receptor genes contain an intron within the protein-coding block.

DNA blot analysis of the cloned rat beta 3-adrenergic receptor gene revealed unexpected restriction enzyme cleavage sites that suggested the presence of one or more introns near the end of the coding block. This region of the rat gene was mapped and sequenced and was found to contain two introns. The first intron occurs 12 amino acids from the end of the coding block, as deduced by comparison with the beta 3 receptor cDNA. Sequence analysis of the first intron indicates that it might contain enhancer elements that could be important in the adipose tissue-specific expression of this gene. The mouse and human beta 3 receptor genes have been assumed to be intronless; however, these genes contain potential splice sites that are homologous to those present in the rat gene. The relevant regions of the mouse and human beta 3 receptor cDNAs were cloned and, by comparing them to the respective genomic sequences, it was concluded that these genes also contain one or more introns. Sequence analysis of the mouse and human beta 3 receptor cDNAs indicates that they code for proteins that are, respectively, 12 and 6 amino acids larger than previously deduced from genomic clones.

Perinatal changes in the coupling of beta 1- and beta 3 adrenergic receptors to brown fat adenylyl cyclase.

The stimulation of adenylyl cyclase by catecholamines in neonatal brown adipose tissue (BAT) is markedly biphasic, suggesting the existence of receptors that have both high and low affinities for catecholamines. The identities of these receptors were examined by comparing responses in neonatal BAT membranes to those of Chinese hamster ovary cells which had been transfected to express the cloned rat beta 1 and beta 3 receptors. The results from these experiments indicate that high-affinity stimulation of adenylyl cyclase by catecholamines in BAT is mediated by beta 1 receptors, as evidenced by the potencies of norepinephrine and isoproterenol at this receptor and the potent blockade of the receptor by alprenolol. The low-affinity catecholamine receptor appears to be the beta 3 receptor, as indicated by the low potency of catecholamine agonists and the inability of low concentrations of alprenolol to block this activity. Furthermore, this receptor, like the cloned rat beta 3 receptor, was antagonized by (-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one (CGP 12177) and was stimulated by (R',R')-4-(2-[(2[(3-chlorophenyl)-2- hydroxyethyl]amino)propyl]phenyl)phenoxyacetic acid (BRL 37344). These results indicate that both beta 1 and beta 3 receptors couple to adenylyl cyclase in BAT and that activation of adenylyl cyclase in neonatal BAT is mediated primarily by beta 3 receptors. Beta 3 receptors were also clearly detected in weanling BAT with the beta 3-selective agonist BRL 37344. However, when catecholamines were used to stimulate activity, the activation of adenylyl cyclase by beta 1 receptors, which occurred at low concentrations of catecholamines, obscured the activation of adenylyl cyclase by beta 3 receptors, which occurred only at high concentrations.

Differential adrenergic regulation of beta 1- and beta 3-adrenoreceptor messenger ribonucleic acids in adipose tissues.

The levels of beta 1- and beta 3-adrenoreceptor mRNAs in several rat tissues were determined simultaneously with a sensitive nuclease protection assay. The beta 1-receptor gene was expressed to varying degrees in most tissues examined. By contrast, high levels of beta 3-receptor mRNA were only found in brown and white adipose tissues, indicating that beta 3-receptor gene expression is essentially adipose tissue specific. Surgical sympathectomy of interscapular brown adipose tissue increased beta 3-receptor mRNA levels by 2.4-fold, but did not affect beta 1-receptor mRNA levels. Exposure of rats to 4 C, which increases sympathetic nerve stimulation of IBAT, reduced beta 3-receptor mRNA levels in intact tissue but did not affect the denervation-induced increase in beta 3-receptor mRNA. Acute treatment of rats with norepinephrine greatly reduced beta 3 mRNA levels in both white and brown adipose tissues, but did not alter beta 1-receptor mRNA levels. These results indicate that beta 1- and beta 3-receptor mRNAs are differentially regulated and that norepinephrine released from sympathetic nerves is an important inhibitory regulator of beta 3-receptor mRNA levels. Injections of the beta-receptor agonist isoproterenol and the beta 3-receptor agonist BRL 26830 each reduced beta 3-receptor mRNA in brown and white fat, whereas injections of glucagon reduced beta 3-receptor mRNA in brown fat only. These data indicate that while stimulation of beta 3-receptors is sufficient to down-regulate beta 3 mRNA, other receptors that stimulate adenylyl cyclase have the same effect. Finally, the agonist-induced down-regulation of beta 3-receptor mRNA was associated with a reduction in beta 3-receptor activation of adenylyl cyclase in white adipose tissue.

Molecular cloning and expression of the rat beta 3-adrenergic receptor.

Rat adipose tissues contain atypical beta receptors that display certain pharmacological sensitivities that are similar to those found in the recently cloned human beta 3 receptor. However, there are also certain pharmacological differences between the human atypical beta 3 receptor and atypical receptors in rodent adipose tissues, which could indicate strong species differences, the existence of multiple atypical receptor subtypes, or both. To help decide among these possibilities, a rat beta 3 receptor clone was obtained and expressed in Chinese hamster ovary cells. The predicted primary structures of the rat and human receptors are greater than 90% similar. Despite this similarity, the pharmacological properties of the rat receptor differed from those reported for the human receptor but were similar to the properties exhibited by atypical receptors in rat adipose tissue. Specifically, the rat beta 3 receptor had a high affinity for BRL 37344 and a relatively low affinity for norepinephrine and was partially activated by the beta 1 and beta 2 receptor antagonist CGP 12177. Northern blot analysis and nuclease protection assays of RNA from rat tissues indicate that the beta 3 receptor is abundantly expressed only in adipose tissues.

Nitrate effects on nitrate reductase activity and nitrite reductase mRNA levels in maize suspension cultures.

Nitrate reductase (NR) activity and nitrite reductase (NiR) mRNA levels were monitored in Black Mexican Sweet maize (Zea mays L.) suspension cultures after the addition of nitrate. Maximal induction occurred with 20 millimolar nitrate and within 2 hours. Both NR and NiR mRNA were transiently induced with levels decreasing after the 2 hours despite the continued presence of nitrate in the medium. Neither ammonia nor chlorate prevented the induction of NR. Furthermore, removal of nitrate, followed by its readdition 22 to 48 hours later, did not result in reinduction of activity or message. NR was synthesized de novo, since cycloheximide completely blocked its induction. Cycloheximide had no effect on the induction of NiR mRNA or on the transient nature of its induction. These results are similar to those reported previously for maize seedlings.

Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation.

We have constructed a set of plant transformation vectors, promoter cassettes, and chimeric antibiotic-resistance genes for the transformation and expression of foreign genes in plants sensitive to Agrobacterium infection. The different vectors allow for either concurrent or consecutive selection for kanamycin and hygromycin resistance and have a number of unique restriction sites for the insertion of additional DNA. The promoter cassettes utilize the CaMV 19S and CaMV 35S promoters and are constructed to allow for the easy insertion of foreign genes. The cloned gene can then easily be inserted into the transformation vectors. We have utilized the promoter cassettes to express the hygromycin-resistance gene either from the CaMV 35S or the CaMV 19S promoters, with both chimeric resistance genes allowing for the selection of hygromycin-resistant tobacco plants.

Synthesis and secretion of wheat alpha-amylase in Saccharomyces cerevisiae.

A wheat alpha-amylase cDNA clone has been fused to the phosphoglycerate kinase initiator methionine to enable synthesis in the yeast Saccharomyces cerevisiae of an alpha-amylase enzyme that is identical in size to the wild-type alpha-amylase. The alpha-amylase is synthesized with an N-terminal plant signal peptide which is recognized in the yeast host, leading to efficient processing and secretion into the medium. The secretion of alpha-amylase into the medium is quite efficient in rich medium, but barely detectable in a minimal medium.