Agonist-mediated down-regulation of rat beta1-adrenergic receptor transcripts: role of potential post-transcriptional degradation factors.

The human beta1-adrenergic receptor (AR) and hamster beta2-AR transcripts can be post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of their 3' untranslated regions (UTR) with RNA binding proteins. Using RNase protection assays, we have determined that chronic isoproterenol exposure of rat C6 glioma cells results in the accelerated reduction of beta1-AR mRNAs. To determine the role of cellular environment on the agonist-independent and agonist-mediated degradation of beta1-AR mRNAs, we transfected rat beta1-AR expression recombinants into both hamster DDT1MF2 cells and rat L6 cells. The rat beta1-AR mRNAs in the two transfectant cell pools retain longer agonist-independent half-lives than in the C6 environment and undergo accelerated degradation upon chronic agonist exposure. Using UV-cross-linking/immunoblot and immunoprecipitation analyses, we have determined that the rat beta1-AR 3' UTR recognizes a predominant M(r) 39,000 component, identified as the mammalian elav-like protein HuR, and several other minor components, including the heteronuclear protein hnRNP A1. HuR levels are more highly expressed in C6 cells than in DDT1MF2 and L6 cells and are induced after chronic isoproterenol treatment. Furthermore, C6 transfectants containing an HuR expression recombinant exhibit reduced beta1-AR mRNA half-lives that were statistically comparable with half-lives identified in isoproterenol-treated C6 cells. These results imply that HuR plays a potential role in the agonist-independent and agonist-mediated down-regulation of beta1-AR mRNAs.

Simian retrovirus vectors for gene transfer in nonhuman primate cells.

We have recently identified and sequenced a molecular clone of the serogroup 2 simian retrovirus (SRV), D2/RHE/OR/V1, that retains an enhanced ability to infect specific T cell lines. In this report, using deletion mutagenesis, we localized the psi packaging signal, necessary for packaging of D2/RHE/OR/V1 particles, to the genomic region 345-650, which comprises the 5' intergenic region (IR) and the extreme 5' portion of the gag gene. To build an SRV-based gene transfer system and to reduce the possibility of recombination and regeneration of replication-competent viruses, we constructed split-genome D2/RHE/OR/V1 plasmid recombinants containing distinct and non-overlapping retroviral gene regions and several replacement components. For the retrovirus gene transfer vehicle, we deleted the D2/RHE/OR/V1 structural genes and substituted a cassette including the psi-packaging region, the beta-galactosidase reporter gene, and the 3' IR. Both packaging cell recombinants were used to generate stable monkey packaging cell lines; the gene transfer vehicle was subsequently transfected into the packaging cell lines, and replication-defective viruses were recovered for subsequent infection into fresh monkey cells. Successful infection by the recovered viruses verifies the potential efficacy of the SRV-based system as a research tool for gene transfer of heterologous genes into nonhuman primate cells.

Simian retrovirus serogroup 5: partial gag-prt sequence and viral RNA distribution in an infected rhesus macaque.

The simian type D retroviruses (SRVs) are one of the causative agents of simian acquired immunodeficiency syndrome (SAIDS) in Asian macaques. In this report, we describe the infection of a rhesus macaque with the SRV serogroup 5 isolate, D5/RHE/OR. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and dot blot hybridization analyses, we have determined the tissue distribution of D5/RHE/OR in this infected rhesus macaque, and have demonstrated viral mRNA in the majority of the surveyed tissues, including robust loads in the bone marrow, seminal vesicle, submaxillary salivary gland, prostate, and skeletal muscle. Microscopic examination of necropsy tissues revealed generalized lymphoid hyperplasia that was most severe in the salivary gland, bone marrow, kidney, and spleen. We also describe the first sequence analyses of portions of the D5/RHE/OR gag-prt region, obtained as a RT-PCR amplification product from infected rhesus macaque tissue, and report the first confirmation using Northern blot analyses that the SRV serogroups, including D5/RHE/OR, express similarly-sized genomic and subgenomic env mRNAs.

Rat beta 1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins.

beta 1-Adrenergic receptors (beta1-ARs) serve as important regulators of central nervous system (CNS)-mediated behavior and several neural functions, including mood, memory, neuroendocrine control, and stimulation of autonomic function. Using beta 1-AR-luciferase reporter recombinants, we have previously determined that important beta 1-AR genetic elements controlling expression within the C6 glioma cell line are contained within the region -396 to -299, relative to the translational start site. By conducting progressive internal deletions of the rat beta 1-AR 5' flanking region and with the use of beta 1-AR-luciferase recombinants, we have verified that this region contains the primary beta 1-AR promoter and/or major regulatory elements. To begin the identification of protein factors involved in beta 1-AR transcriptional activity conferred by this beta 1-AR region and flanking sequences, we conducted electrophoretic mobility shift assays using defined beta 1-AR DNA subregion probes. One probe (GS-1), encompassing the region -396 to -367, was found to produce two major and two minor mobility shift complexes when bound to nuclear extracts from the beta 1-AR expresser C6 cell line. UV-crosslinking of DNA-protein complexes, coupled with DNase I digestion, indicated that this beta 1-AR region interacts with one major protein of approximately 117 kDa molecular weight and additional minor proteins. GS-1 DNA-protein complexes were observed using beta 1-AR expresser tissues in the CNS, including cortex, hippocampus, and olfactory bulb. No DNA-protein complexes were observed when using nuclear extracts from beta 1-AR nonexpresser tissues; in some cases, using L6 cells, previously characterized to express little or no beta1-ARs, a reduction in intensities of the DNA-protein complexes was observed. Competition experiments indicate that nuclear protein binds to one of two subregions within the GS-1 sequence that contain AP-2-like consensus elements. Recombinant AP-2 protein will bind to both the beta 1-AR GS-1 promoter fragment and commercially available AP-2 consensus element control probes. Interestingly, using antibody supershift and immunoblotting experiments, no supershifts were observed and the major 117-kDa protein was not immunoreactive to antibodies recognizing either AP-2 alpha or AP-2 beta. These results support our contention that this beta 1-AR regulatory region contains AP-2 consensus elements that recognize novel transactivator proteins.

Nucleocytoplasmic export of type D simian retrovirus genomic RNA: identification of important genetic subregions and interacting cellular proteins.

The simian retrovirus (SRV) genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. The serogroup 2 SRV CTE is predicted to form a stable stem-loop structure containing two major internal loops exhibiting 180 degrees inverse symmetry, with loop face sequences A, A', B, and B' and additional minor internal and terminal loops. To begin the identification of potential CTE-interacting proteins and to assess structural requirements for protein interaction, we conducted RNA mobility shift assays using IR fragments that obliterated this region's known stable stem-loop structure. Using immunoblotting assays, we have determined that RNA helicase A, implicated in the nuclear export of unspliced SRV genomic RNA, does not appear to interact directly with either the complete serogroup 2 SRV 3' IR or the subregion RNAs and that formation of RNA-protein complexes is conferred by interaction with other novel proteins. UV crosslinking of RNA-protein complexes, coupled with RNase T1/A digestion, indicates that a novel protein of 120 kDa molecular weight interacts with the complete CTE or with individual subregion RNAs. Transfection analyses indicate that SRV recombinants containing A, A', B, or B' sequences forming the faces for two open loops undergo RNA export; only the complete sense CTE recombinant or a second recombinant containing two subregions in sense orientation that reconstitute the 3' two-thirds of the 3' IR, and contain only A' and B that form the faces for two terminal loops, are capable of SRV RNA export. These experiments indicate that secondary structural determinants of the 3' IR and multiple protein interactions may be important factors in the nuclear export of unspliced SRV RNA.

Molecular cloning and cell-specific growth characterization of polymorphic variants of type D serogroup 2 simian retroviruses.

Simian retroviruses (SRVs), the etiological agent of a spontaneous Simian acquired immunodeficiency syndrome, endemically infects large percentages of Asian macaques housed in biomedical research colonies and severely compromises the effective use of these species as a viable research animal. We recently described the molecular cloning of a serogroup 2 SRV, D2/RHE/OR, which causes mild immunosuppression in rhesus macaques. A restriction site variant, D2/RHE/OR/V1, has also been recovered from severely ill animals endemically infected with D2/RHE/OR. We now report the complete nucleotide sequences of D2/RHE/OR and D2/RHE/OR/V1. Both infectious molecular clones retain the genetic structure typical of type D SRVs (5' LTR-gag-prt-pol-env-3'LTR) and encode identically sized 8105-bp proviruses. D2/RHE/OR and D2/RHE/OR/V1 are 99.3% similar at the amino acid level, exhibiting only 17 residue differences, of which 10 are located in the envelope glycoproteins. The molecular clones and reciprocal chimeric viruses were used to assess the contribution of different genetic domains to virus infectivity in a T cell infection assay. These experiments indicate that D2/RHE/OR has a reduced ability to infect specific T cell lines, especially Hut-78 and MT-4 cells, and that the envelope gene is not the sole determinant of in vitro tropism.

Protein kinase C-mediated down-regulation of beta1-adrenergic receptor gene expression in rat C6 glioma cells.

In the current study, we investigated the mechanism by which protein kinase C (PKC) regulates the expression of beta1-adrenergic receptor (beta1AR) mRNA in rat C6 glioma cells. Exposure of the cells to 4beta-phorbol-12-myristate-13-acetate (PMA), an activator PKC, resulted in a down-regulation of both beta1AR binding sites and mRNA levels in a time- and concentration-dependent manner. This effect was not observed with phorbol esters that do not activate PKC and was blocked by bisindolylmaleimide, a specific PKC inhibitor. Activation of PKC did not reduce the half-life of beta1AR mRNA but significantly decreased the activity of the beta1AR promoter, as determined by reporter analysis. A putative response element, with partial homology to a consensus cAMP response element, was identified by mutation analysis of the promoter at positions -343 to -336, relative to the translational start site. Mutation of this putative regulatory element, referred to as a beta1AR-PKC response element, completely blocked the PKC-mediated down-regulation of beta1AR promoter activity. Gel mobility shift analysis detected two specific bands when C6 cell extracts were incubated with a labeled DNA probe containing the beta1AR-PKC response element sequence. Formation of one of these bands was inhibited by an oligonucleotide probe containing a consensus CRE and disrupted by an antibody for cAMP response element binding protein. Based on these studies, we propose that the PKC-induced down-regulation of beta1AR gene transcription in C6 cells is mediated in part by a cAMP response element binding protein-dependent mechanism acting on a novel response element.

Adrenergic regulation of ICER (inducible cyclic AMP early repressor) and beta1-adrenergic receptor gene expression in C6 glioma cells.

ICER (inducible cyclic AMP early repressor), a member of the cyclic AMP response element (CRE) modulator (CREM) family of transcription factors, is a powerful repressor of cyclic AMP-mediated transactivation. Our studies characterize the regulation of ICER in C6 glioma cells and investigate its role in repressing transcription of the beta1-adrenergic receptor (beta1AR) gene. Incubation with isoproterenol (100 nM) results in a rapid induction in levels of mRNA for ICER and its splice variant ICERgamma, with maximal induction occurring after 2 h of treatment. Incubation with isoproterenol also increased levels of CREM isoforms within 1 h; this was unexpected given previous reports that these isoforms are not rapidly induced. Increased expression of ICER and CREM was accompanied by induction of two CRE-binding complexes. The presence of ICER in these two CRE-binding complexes is demonstrated by their disruption with CREM antibody and by their comigration with recombinant ICER. Because the time course for isoproterenol induction of ICER mRNA and CRE binding corresponds to that for down-regulation of beta1AR mRNA levels in C6 glioma cells, the influence of ICER beta1AR transcription was directly examined. Coexpression of ICER significantly decreased transcriptional activity of a rat beta1AR promoter-luciferase reporter construct that contains a CRE. In contrast, coexpression of ICER did not influence two truncated rat beta1AR promoter constructs that lack the CRE site. These data demonstrate that ICER can interact at the beta1AR promoter to repress transcription.

Genetically modified PC12 brain grafts: survivability and inducible nerve growth factor expression.

Neural transplantation of genetically modified cells has been successfully employed to reverse functional deficits in animal models of neurodegenerative disorders, including Parkinson's disease. While implanted PC12 cells secrete dopamine in vivo and can ameliorate dopamine deficiency in parkinsonian rat model systems, these cells either degenerate within 2-3 wk postimplantation (presumably due to the lack of neural trophic factor support at the site of implantation), or in some cases, form a tumor mass leading to the death of the host animal. To address these limitations, we have developed a genetically modified PC12 cell line that can synthesize nerve growth factor (NGF) under the control of a zinc-inducible metallothionein promoter. When implanted in the rat striatum and under in vivo zinc stimulation, these cells will neuro-differentiate, express tyrosine hydroxylase, and will undergo survival through potential autocrine trophic support. This regulatable cell line and general approach may provide additional insight on the potential utilization of cell transplants for treatment of Parkinson's disease and other neurodegenerative disorders.

Distribution of dopamine D1, D2, and D5 receptor mRNAs in the monkey brain: ribonuclease protection assay analysis.

The distribution of the mRNAs encoding the dopamine D1, D2 and D5 receptors was determined in brain tissues obtained from intact female rhesus monkeys, using a ribonuclease protection assay. Tissue blocks from the frontal cortex, striatum, thalamus, hippocampus and substantia nigra were dissected and total RNA was extracted. Dopamine D2 and D5 receptor DNA fragments were generated from rhesus monkey genomic DNA using polymerase chain reaction. To generate dopamine receptor subtype-specific cRNA probes, DNA fragments corresponding to the carboxy terminus of the rhesus monkey D1 and D2 receptor genes and to the putative transmembrane domain regions (IV-VI) of the D5 receptor gene, were subcloned into the pGEM3Z/4Z vectors. Expression of D1 receptor mRNA exhibited significant regional differences: striatum > > > cerebral cortex > or = hippocampus > or = lateral thalamus. D1 receptor mRNA was found in low quantities in the medial thalamus, but was not consistently expressed in the substantia nigra area. In contrast, D2 receptor mRNA was detected in all regions that were studied: striatum > > > substantia nigra > > hippocampus > or = cerebral cortex > or = medial thalamus > or = lateral thalamus. D5 receptor mRNA was also expressed in all regions, with highest levels in the cerebral cortex, striatum and lateral thalamus, and moderate levels in the substantia nigra, medial thalamus and the hippocampus. The D5 receptor mRNA appears to be widely distributed in the monkey brain. Most interesting is the expression of D5 receptor mRNA in tissues of the substantia nigra area.

Transcription of the rat beta 1-adrenergic receptor gene. Characterization of the transcript and identification of important sequences.

We have characterized the 5' and 3' ends of the rat beta 1-adrenergic receptor transcript using RNase protection assays and have used transient transfection analysis to identify regions of the beta 1-adrenergic gene 5'-flanking sequences which are important for expression. The transcript has multiple start sites, occurring primarily in two clusters at bases -250 and -280, relative to the first base of the initiation codon. Two potential polyadenylation signals at +2450 and +2732 are both functional, although the site at +2732 is preferred both in C6 glioma cells and in heart tissue. Characterization of the gene by transient transfection analysis has identified a region between bases -389 and -325 which is necessary for expression. The specific deletion of a potentially functional inverted CCAAT sequence within this region does not significantly alter activity. In addition to the region from -389 and -325, deletion of the bases between -1 and -159 and between -186 and -211 significantly alters expression. Both of these regions are down-stream from the beta 1-adrenergic receptor gene start sites and may function either through regulation of transcription or through alteration of the transcript structure.

Chimeric D1/D2 dopamine receptors. Distinct determinants of selective efficacy, potency, and signal transduction.

D1/D2 chimeras were constructed that had D1 dopamine receptor sequence at the amino-terminal end and D2 dopamine receptor sequence at the carboxyl-terminal end. The chimeras with the first four, five and six transmembrane domains of the D1 receptor (CH2, CH3, CH4, respectively) bound the D1 receptor antagonist [3H]SCH 23390 with high affinity. Reciprocal chimeras constructed with D2 receptor sequence at the amino-terminal end displayed no detectable specific binding of [3H]SCH 23390, [125I]epidepride, or [3H]spiperone. CH2, CH3, and CH4 had lower affinity than either D1 or D2 dopamine receptors for the nonselective antagonists and agonists and D2-selective antagonists tested. The chimeric receptors had affinities for three D1-selective ligands and the D2-selective agonist, quinpirole, that were intermediate between D1 and D2 receptor affinities for the drugs. The substantial loss or gain of affinity for three ligands upon replacement of D1 transmembrane VII with D2 sequence (CH4) suggests an important role for this region in the selectivity of these drugs. Stimulation of adenylyl cyclase activity by D1 agonists occurred in cells expressing CH3 and CH4, both of which included the D1 third cytoplasmic loop, but not in cells expressing CH1 or CH2, both with the D2 third cytoplasmic loop. However, only CH3 was able to mediate stimulation of adenylyl cyclase by quinpirole, implying that D2 receptor transmembrane domain VI was an important determinant of the selective efficacy of quinpirole. On the other hand, transmembrane domain VII was particularly important for the selective potency of quinpirole. Inhibition of beta-adrenergic receptor-stimulated adenylyl cyclase activity by dopamine was seen in cells expressing D2 receptors and CH1, but not CH2, CH3, or CH4. Thus, the third cytoplasmic loop of D1 dopamine receptors was crucial for the coupling of the receptors to Gs, but inhibition of adenylyl cyclase via Gi required structural features, such as the second cytoplasmic loop of the D2 receptor, in addition to the 3rd cytoplasmic loop.

Polymerase chain reaction detection of type D simian retrovirus proviral DNA from infected macaques.

A simple polymerase chain reaction (PCR) approach was developed for detection of Type D simian retrovirus (SRV) serogroup 2 proviral DNA using peripheral blood lymphocytes (PBLs) obtained from infected macaques. PCR primer pairs were developed against serogroup 2 envelope (env) gene sequence, and fidelity of PCR fragment amplification was determined using molecularly cloned SRV serogroup 2 (D2/RHE/OR) DNA, and genomic DNA from Raji cells independently infected with different SRV serogroups. One primer pair exhibiting high fidelity was then utilized for PCR detection of serogroup 2 proviral DNA from PBLs, and from cells sorted into immune cell subpopulations by fluorescent-activated cell sorting (FACS). Env PCR fragments were readily detected from as few as 10(4) PBLs or immune cell subpopulations. In addition, highly specific PCR primers against serogroups 1 and 3 were utilized to detect proviral DNA from Raji cells infected with SRV serogroups. In all cases, primers designed to amplify serogroups 1, 2, and 3 proviral DNA were specific for their intended serogroup. This primer information and development of a PCR approach for detection of specific SRV proviral DNA will be of potential utility as a rapid surveillance tool in monitoring type D simian retrovirus infection within Asian macaque colonies.

The 5' flanking region of the rat beta 3-adrenergic receptor gene: divergence with the human gene and implications for species-specific gene expression.

beta 3-adrenergic receptor mRNAs exhibit species-specific expression (human vs. rodent) in distinct anatomical regions and appear to be expressed abundantly within rodent adipose tissue, but only at low levels within corresponding human tissues. In order to determine the genetic basis of the differential expression of the rat and human beta 3-adrenergic receptor genes, we cloned and sequence the rat gene and compared the 5' flanking regions of the two genes to identify potential discriminators in transcriptional regulation. We have found that the rat and human beta 3-adrenergic receptor 5' flanking regions are only 67% similar, unlike the close sequence similarity observed between the coding blocks (> 90%) and also observed between species for the 5' flanking regions of other beta-adrenergic receptor subtype genes (> 90%). In addition, the rat beta 3-adrenergic receptor gene lacks the four potential cAMP responsive elements identified within the 5' flanking region of the human receptor gene. The striking divergence in regulatory sequences between the rat and human beta 3-adrenergic receptor genes may potentially explain the differences in species-specific expression and tissue localization of the rat and human receptor mRNAs.

The rhesus macaque beta 1-adrenergic receptor gene: structure of the gene and comparison of the flanking sequences with the rat beta 1-adrenergic receptor gene.

We have cloned the gene for the rhesus macaque beta 1-adrenergic receptor. In addition to the protein coding block, we have sequenced its 5' (1424 bp) and 3' (1534 bp) flanking regions and aligned them with comparable sections of the rat beta 1-adrenergic receptor gene. The rhesus macaque gene contains a 1440 bp open reading frame which codes for a deduced protein of 480 amino acids that is 95% and 89% similar to the human and rat beta 1-adrenergic receptors, respectively. The rhesus macaque beta 1-adrenergic receptor contains conserved sites for potential N-linked glycosylation and cAMP-dependent protein kinase phosphorylation identified within the human and rat receptors, but differs in the structure and length of the third cytoplasmic loop. The 400 bases of 5' flanking sequence proximal to the protein coding block are highly conserved (84% similarity) between the rat and rhesus macaque genes. The entire 3' flanking sequence, which extends beyond two potential polyadenylation sites at 1050 and 1337 bp relative to the translation termination codon, is also highly conserved between the two species. Comparison of the flanking sequences of the two species reveals conserved regulatory sequences which may be important for beta 1-adrenergic receptor expression and transcriptional modulation.

Decreased accumulation of beta 1-adrenergic receptor, G alpha s and total myosin heavy chain messenger RNAs in the left ventricle of senescent rat heart.

The expression of genes coding for the beta 1-adrenergic receptor (beta 1-AR), the alpha subunit of Gs and total myosin heavy chain (MHC) was compared between left ventricles (LV's) from young (6-7 weeks old) and old (22 months old) rats. The mRNA levels were quantitated by Northern or Slot blots analyses using specific DNA probes. Ageing was found to be associated with a reduction in beta 1-AR (77%), G alpha s (33%) and, total MHC (51%) mRNA levels with no concomitant change in 18S RNA and poly(A+) mRNA levels. These results indicate that transcriptional and/or post-transcriptional mechanisms participate in the control of beta-adrenergic receptor density during ageing. As in the senescent LV, beta 1-AR mRNA level is reduced in the hypertrophied LV, whereas the level of G alpha s mRNA is reduced in the senescent but not in the hypertrophied LV. From our data we conclude (1) that a dual mechanism may operate during ageing, mechanical factors indirectly regulating beta 1-AR mRNA level, while changes in G alpha s mRNA level do not depend on hemodynamic load and (2) that the re-expression of beta-MHC mRNA does not compensate for the decreased accumulation of alpha-MHC mRNA which results in a large decrease in the level of total MHC mRNA in the senescent LV.

Beta 1 adrenergic receptor and G alpha s mRNAs in rat heart as a function of mechanical load and thyroxine intoxication.

OBJECTIVE: The aim of this study was to determine the expression of genes coding for the beta 1 adrenergic receptor and the alpha subunit of Gs in the adult rat normal and hypertrophied left ventricle, and in the left ventricle of the hypophysectomised rat after T4 intoxication. METHODS: Total RNA was extracted from normal, control, or hypertrophied left ventricles 5 weeks after aortic stenosis, and from left ventricles of control or T4 injected hypophysectomised animals. The expression of beta 1 adrenergic receptor and G alpha s mRNAs was quantitated by northern blot analysis and hybridisation with specific 32P-dCTP labelled DNA probes. RESULTS: beta 1 Adrenergic receptor mRNA was decreased (by 33%) in compensated left ventricular hypertrophy without modification of the relative level of G alpha s mRNA. The relative level of beta 1 adrenergic receptor mRNA correlated negatively with the degree of left ventricular hypertrophy, suggesting that the expression of the beta 1 adrenergic receptor gene is not activated by pressure overload. In the left ventricle of the hypophysectomised rat, a rapid increase in beta 1 adrenergic receptor mRNA (by 180% 3 h after hormone injection) was observed in response to T4, with no change in the relative content of G alpha s mRNA. These results provide evidence that beta 1 adrenergic receptor mRNA and G alpha s mRNA accumulate to different levels of abundance in the adult left ventricle, as indicated by their ratios (0.053 and 0.043 in sham operated and hypertrophied left ventricles respectively). This suggests that distinct mechanisms are involved in the control of the accumulation of these two mRNAs in cardiac tissue. CONCLUSIONS: The reduction in beta 1 adrenergic receptor density in the hypertrophied rat left ventricle is associated with a parallel reduction in the level of beta 1 adrenergic receptor mRNA. The beta 1 adrenergic receptor gene may belong to a group of genes which are not activated by pressure overload, but are responsive to thyroid hormone.