The cellular localization, agonist-mediated internalization, and desensitization properties of the alpha(1)-adrenoceptor (alpha(1)-AR) subtypes conjugated with green fluorescent protein (alpha(1)-AR/GFP) were assessed using real-time imaging of living, transiently transfected human embryonic kidney (HEK) 293 cells. The alpha(1B)-AR/GFP fluorescence was detected predominantly on the cell surface. Stimulation of the alpha(1B)-AR with phenylephrine led to an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and promoted rapid alpha(1B)-AR/GFP internalization. Long-term exposure (15 h) to phenylephrine resulted in desensitization of the alpha(1B)-AR-mediated activation of ERK1/2 phosphorylation. Alpha(1A)-AR/GFP fluorescence was detected not only on the cell surface but also intracellularly. The rate of internalization of the cell surface population alpha(1A)-AR/GFPs was slower than that seen for the alpha(1B)-AR. Agonist exposure also resulted in desensitization of the alpha(1A)-AR-mediated increase in ERK1/2 phosphorylation. The alpha(1D)-AR/GFP fluorescence was detected mainly intracellularly, and this localization was unaffected by exposure to phenylephrine. Phenylephrine treatment of alpha(1D)-AR/GFP expressing cells increased ERK1/2 phosphorylation. However, this increase was not significant. Cotransfection with beta-arrestin 1 did not increase the rate or extent of agonist-stimulated alpha(1A)- or alpha(1B)-AR/GFP internalization. However, a dominant-negative form of the beta-arrestin 1, beta-arrestin 1 (319-418), blocked agonist-mediated internalization of both the alpha(1A)- and alpha(1B)-ARs. These data show that transfected alpha(1)-AR/GFP fusion proteins are functional, that there are differences in the cellular distribution and agonist-mediated internalization between the alpha(1)-ARs, and that agonist-mediated alpha(1)-AR internalization is dependent on arrestins and can be desensitized by long-term exposure to an agonist. These differences could contribute to the diversity in physiologic responses regulated by the alpha(1)-ARs.
The regulation of the cellular distribution and intracellular signaling properties of the alpha(1B)- and alpha(1D)- adrenoceptor (alpha(1)-AR) subtypes was examined in stably transfected Rat 1 fibroblasts. In unstimulated cells, alpha(1B)-AR expression was noted primarily on the cell surface. Treatment with phenylephrine induced internalization of the alpha(1B)-AR and promoted association with arrestin 2. The internalized alpha(1B)-AR colocalized with the transferrin receptor, an endosomal marker. In unstimulated fibroblasts, the alpha(1D)-AR was detected in a perinuclear orientation and was colocalized with arrestin 2 in a compartment also containing the transferrin receptor. After treatment with prazosin, which exhibits inverse agonist properties, the alpha(1D)-AR was redistributed from intracellular sites to the cellular periphery and was no longer associated with the transferrin receptor or arrestin 2. alpha(1D)-AR-expressing cells exhibited a high degree of basal activity for both inositol phosphate formation and extracellular signal regulated kinase (ERK), which was reduced by treatment with prazosin. In these cells, phenylephrine induced a dose-dependent increase in inositol phosphate formation but had no effect on ERK activity. In alpha(1B) -AR-expressing cells, phenylephrine stimulated both inositol phosphate formation and ERK activity. These data show that: 1) there are differences in the cellular localization of the alpha(1)-AR subtypes; 2) the alpha(1B)-AR exhibits expected G protein-coupled receptor activity regarding cellular localization, agonist-mediated internalization, and coupling to second messengers; and 3) the alpha(1D)-AR is constitutively active and, as a result, is localized to intracellular compartments involved in receptor recycling.
alpha(1)-Adrenergic receptors (ARs) are not well defined in the central nervous system. The particular cell types and areas that express these receptors are uncertain because of the lack of high avidity antibodies and selective ligands. We have developed transgenic mice that either systemically overexpress the human alpha(1A)-AR subtype fused with the enhanced green fluorescent protein (EGFP) or express the EGFP protein alone under the control of the mouse alpha(1A)-AR promoter. We confirm our transgenic model against the alpha(1A)-AR knockout mouse, which expresses the LacZ gene in place of the coding region for the alpha(1A)-AR. By using these models, we have now determined cellular localization of the alpha(1A)-AR in the brain, at the protein level. The alpha(1A)-AR or the EGFP protein is expressed prominently in neuronal cells in the cerebral cortex, hippocampus, hypothalamus, midbrain, pontine olivary nuclei, trigeminal nuclei, cerebellum, and spinal cord. The types of neurons were diverse, and the alpha(1A)-AR colocalized with markers for glutamic acid decarboxylase (GAD), gamma-aminobutyric acid (GABA), and N-methyl-D-aspartate (NMDA) receptors. Recordings from alpha(1A)-AR EGFP-expressing cells in the stratum oriens of the hippocampal CA1 region confirmed that these cells were interneurons. We could not detect expression of the alpha(1A)-AR in mature astrocytes, oligodendrocytes, or cerebral blood vessels, but we could detect the alpha(1A)-AR in oligodendrocyte progenitors. We conclude that the alpha(1A)-AR is abundant in the brain, expressed in various types of neurons, and may regulate the function of oligodendrocyte progenitors, interneurons, GABA, and NMDA receptor containing neurons.
These data demonstrate that alpha1A-ARs protect the heart from ischemic injury through a staurosporine-sensitive signaling pathway that is independent of protein kinase C.
alpha1-Adrenergic receptors (ARs) are well-known mediators of the sympathetic nervous system, are highly abundant in the brain, but are the least understood in the central nervous system. The particular cell types in the brain that contain these receptors or their functions are not known because of the lack of high avidity antibodies and selective ligands. We developed transgenic mice that endogenously overexpress the alpha1B-AR subtype fused with the enhanced green fluorescent protein (EGFP). Endogenous expression was obtained by using a 3.4 kb fragment of the mouse alpha1B-AR promoter. Using this model, we determined cellular localization of the alpha1B-AR throughout the brain. The alpha1B-AR-EGFP fusion protein is expressed in neurons throughout the brain and in the Purkinje cells of the cerebellum. The alpha1B-AR is also expressed in NG2 oligodendrocyte precursor cells in both neonatal cell cultures and in the adult cerebral cortex, but is weakly expressed in mature oligodendrocytes. The alpha1B-AR was not observed in astrocytes or in cerebral vascular smooth muscle, cell types previously suggested to contain alpha1-ARs. We conclude that the alpha1B-AR is highly abundant throughout the brain, predominately in neurons, and may be involved in the development of the oligodendrocyte. In adult NG2 cells, implicated in stem cell-like functions, the alpha1B-AR may also play a role. This is the first report of a transgenic tagged-GPCR approach to determine in vivo localization of a receptor.
The regulation of cardiac and vascular function by the ␣ 1B -and ␣ 1D -adrenoceptors (ARs) has been assessed in two lines of transgenic mice, one over-expressing a constitutively active ␣ 1B -AR mutation (␣ 1B -AR C128F ) and the other an ␣ 1D -AR knockout line. The advantage of using mice expressing a constitutively active ␣ 1B -AR is that the receptor is tonically active, thus avoiding the use of nonselective agonists that can activate all subtypes. In hearts from animals expressing ␣ 1B -AR C128F , the activities of the mitogen-activated protein kinases, extracellular signal-regulated kinase, and c-Jun N-terminal kinase were significantly elevated compared with nontransgenic control animals. Mice over-expressing the ␣ 1B -AR C128F had echocardiographic evidence of contractile dysfunction and increases in chamber dimensions. In isolated-perfused hearts or left ventricular slices from ␣ 1B -AR C128F -expressing animals, the ability of isoproterenol to increase contractile force or increase cAMP levels was significantly decreased. In contrast to the prominent effects on the heart, constitutive activation of the ␣ 1B -AR had little effect on the ability of phenylephrine to induce vascular smooth muscle contraction in the isolated aorta. The ability of phenylephrine to stimulate coronary vasoconstriction was diminished in ␣ 1D -AR knockout mice. In ␣ 1D -AR knockout animals, no negative effects on cardiac contractile function were noted. These results show that the ␣ 1 -ARs regulate distinctly different physiologic processes. The ␣ 1B -AR appears to be involved in the regulation of cardiac growth and contractile function, whereas the ␣ 1D -AR is coupled to smooth muscle contraction and the regulation of systemic arterial blood pressure.G-protein-coupled receptors comprise about 1% of the human genome and perform vital and diverse roles in the regulation of physiologic processes. One of the members of the G-protein-coupled receptor family is the ␣ 1 -adrenergic receptor (␣ 1 -AR). Three subtypes, the ␣ 1A -, ␣ 1B -, and ␣ 1D -ARs, have been isolated, cloned, and characterized. These receptors are intimately involved in the regulation of peripheral vascular resistance, cardiac function, and vascular and myocardial cell growth (for recent reviews on all aspects of the ␣ 1 -ARs see García-Sá inz et al
Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.
␣ 1 -Adrenoceptor subtypes (␣ 1A -, ␣ 1B -, ␣ 1D -) are known to couple to similar signaling pathways, although differences among the subtypes do exist. As a more sensitive assay, we used oligonucleotide microarrays to identify gene expression changes in Rat-1 fibroblasts stably expressing each individual subtype. We report the gene expressions that change by at least a factor of 2 or more. Gene expression profiles significantly changed equally among all three subtypes, despite the unequal efficacy of the inositol phosphate response. Gene expressions were clustered into cytokines/growth factors, transcription factors, enzymes, and extracellular matrix proteins. There were also a number of individual subtype-specific changes in gene expression, suggesting a link to independent pathways. In addition, all three ␣ 1 -AR subtypes robustly stimulated the transcription of the prohypertrophic cytokine interleukin (IL)-6, but differentially altered members of the IL-6 signaling pathway (gp-130 and STAT3). This was confirmed by measurement of secreted IL-6, activated STAT3, and gp-130 levels. Activation of STAT3 Tyr705 phosphorylation by the ␣ 1 -ARs was not through IL-6 activation but was synergistic with IL-6, suggesting direct effects. Interestingly, ␣ 1B -AR stimulation caused the dimerization-dependent phosphorylation of Tyr705 on STAT3 but did not activate the transcriptional-dependent phosphorylation of Ser727. The ␣ 1B -AR also constitutively down-regulated the protein levels of gp-130. These results suggest that the ␣ 1B -AR has differential effects on the phosphorylation status of the STAT3 pathway and may not be as prohypertrophic as the other two subtypes.
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