Inducible expression of α1B-adrenoceptors in DDT1 MF-2 cells: comparison of receptor density and response

doi:10.1016/0922-4106(95)90108-6

European Journal of Pharmacology: Molecular Pharmacology

Volume 289, Issue 2, 28 April 1995, Pages 305-310

https://doi.org/10.1016/0922-4106(95)90108-6 Get rights and content

Abstract

Hamster DDT₁ MF-2 smooth muscle cells natively express α_1B-adrenoceptors which are linked to phosphatidylinositol hydrolysis. We studied the relationship between α_1B-adrenoceptor density and response in this cell line by stable transfection with an isopropyl-β-D-thiogalactoside (IPTG)-inducible vector (pOPα_1B) containing the hamster α_1B-adrenoceptor cDNA. Transfected cells showed a 2-fold increase in receptor density compared to untransfected cells due to constitutive activity of the uninduced vector. Induction of vector expression caused a time-dependent increase in receptor density, reaching a maximum 8-fold increase after 48 h. Exposure to different concentrations of inducing agent for 16 h caused a graded increase in both receptor density and norepinephrine-stimulated [³H]inositol phosphate (InsP) formation. A linear correlation between receptor density and maximum InsP response was observed. Induction of receptor expression did not alter the potency of norepinephrine in stimulating [³H]InsP formation, suggesting that there was no receptor reserve, even at very high expression levels. This inducible expression system should be useful for relating receptor density and responsiveness, and comparing the coupling efficiency of closely related subtypes in activating different signal transduction mechanisms.

References (20)

L.E. Cornett et al.
Characterization of the α₁-adrenergic receptor subtypes in a smooth muscle cell line
J. Biol. Chem.
(1982)
G. Engel et al.
[¹²⁵I]BE 2254, a new high affinity radioligand for α₁-adrenoceptors
Eur. J. Pharmacol.
(1981)
C. Han et al.
Subtypes of α₁-adrenoceptors in DDT₁ MF-2 and BC3H-1 clonal cell lines
Eur. J. Pharmacol.
(1992)
S. Llahi et al.
α₁-Adrenergic receptor-mediated activation of phospholipase D in rat cerebral cortex
J. Biol. Chem.
(1992)
A. Nelemans et al.
α-Adrenoceptor regulation of inositol phosphates, internal calcium and membrane current in DDT₁ MF-2 smooth muscle cells
Eur. J. Pharmacol.
(1990)
S. Cotecchia et al.
Molecular cloning and expression of the cDNA for the hamster α₁-adrenergic receptor
L.C. DuCoeur et al.
Control of gene expression in eukaryotic cells using the lac repressor system
Strategies in Mol. Biol.
(1992)
T.A. Esbenshade et al.
Comparison of α₁-adrenergic receptor subtypes and signal transduction in SK-N-MC and NB41A3 neuronal cell lines
Mol. Pharmacol.
(1993)
T.A. Esbenshade et al.
α₁-Adrenergic receptor subtypes
Ann. Rev. Pharmacol. Toxicol.
(1994)
T.A. Esbenshade et al.
Increased voltage-dependent calcium influx produced by α_1B-adrenergic receptor activation in rat medullary thyroid carcinoma 6–23 cells
Mol. Pharmacol.
(1994)

There are more references available in the full text version of this article.

Cited by (22)

Selective inhibition of α<inf>1A</inf>-adrenergic receptor signaling by RGS2 association with the receptor third intracellular loop
2005, Journal of Biological Chemistry
Citation Excerpt :
RGS2 Co-localizes at the Plasma Membrane with α1A-ARs in HEK293 Cells—To determine whether RGS2 associates with α1A-ARs in a cellular context, we co-transfected GFP-tagged RGS proteins with HA-tagged α1-ARs into HEK293 cells, and we examined their cellular localization by using confocal microscopy. However, to ensure that these effects are not a result of receptor and/or RGS overexpression, we performed radioligand binding experiments using 125I-BE2254 on cells transiently expressing HA-α1A-AR and HA-α1B-ARs, and we found that both constructs express between 100 and 300 fmol/mg protein (data not shown), which is well within the physiological range for expression of these receptors in native systems (27). In addition, we found that when titrating the concentration of GFP-tagged RGS constructs to be used in transfection from 0.5 to 6 μg of cDNA per 30-mm plate, 3 μg of construct resulted in ∼60% transfection efficiency with the majority of the cells exhibiting low to moderate fluorescence (data not shown).
Regulators of G-protein signaling (RGS) proteins act directly on Gα subunits to increase the rate of GTP hydrolysis and to terminate signaling. However, the mechanisms involved in determining their specificities of action in cells remain unclear. Recent evidence has raised the possibility that RGS proteins may interact directly with G-protein-coupled receptors to modulate their activity. By using biochemical, fluorescent imaging, and functional approaches, we found that RGS2 binds directly and selectively to the third intracellular loop of the α_1A-adrenergic receptor (AR) in vitro, and is recruited by the unstimulated α_1A-AR to the plasma membrane in cells to inhibit receptor and G_q/11 signaling. This interaction was specific, because RGS2 did not interact with the highly homologous α_1B- or α_1D-ARs, and the closely related RGS16 did not interact with any α₁-ARs. The N terminus of RGS2 was required for association with α_1A-ARs and inhibition of signaling, and amino acids Lys²¹⁹, Ser²²⁰, and Arg²³⁸ within the α_1A-AR i3 loop were found to be essential for this interaction. These findings demonstrate that certain RGS proteins can directly interact with preferred G-protein-coupled receptors to modulate their signaling with a high degree of specificity.
α <inf>2B</inf>-Adrenoceptor levels govern agonist and inverse agonist responses in PC12 cells
2003, Biochemical and Biophysical Research Communications
Citation Excerpt :
Despite a 70-fold increase in the density of α1B-AR in CHO cells, NE-induced Ca2+ responses showed similar EC50 values while the maximal response was increased about 6-fold [12]. Increasing α1B-AR expression in DDT1 cells caused a linear increase in the maximal response to NE-stimulated inositol phosphate formation, but the EC50 values remained essentially constant [11]. Our previous work showed that the potency of NE did not change when stimulatory adenylyl cyclase activity was studied in CHO cells with varying densities of α2B-AR expression.
Receptor density is an important determinant of cellular effector responses to receptor activation. We analysed cytosolic Ca²⁺ responses to α₂-adrenergic agents in PC12 cells expressing human α_2B-adrenergic receptors (AR) at two densities (3.8 and 1.3 pmol/mg protein). The efficacy (E_max) of agonists was greater in cells with higher receptor expression; while the potency (EC₅₀) of norepinephrine and oxymetazoline was independent of α_2B-AR levels. Several classical α₂-AR antagonists behaved as either partial or inverse agonists in a receptor density-dependent fashion. No apparent structural similarities were found among the inverse agonists, precluding simple predictions of inverse agonist activity. Transfected PC12 cells expressing α_2B-AR at relatively high density would be a useful approach to screen inverse agonists for this class of receptors. Our results further indicate that receptor density significantly influences the properties of ligands, not only of partial agonists as predicted by classical receptor theory, but also of antagonists and full agonists.
Biochemical and pharmacological control of the multiplicity of coupling at G-protein-coupled receptors
2003, Pharmacology and Therapeutics
Citation Excerpt :
However, this approach has been criticised, as it is demonstrated that the signal complexity depends from the cell line used, which may differ in the expression level of signalling partners (G-proteins, effectors, etc.) (Schneider et al., 1994). Indeed, altering the stoichiometry of receptor, G-proteins, and intracellular effectors directly affects the efficacy and potency of agonists at eliciting cellular responses (Alberts et al., 2000; Burt et al., 1998; Eason & Liggett, 1995; Esbenshade et al., 1995; Grunewald et al., 1996; Kukkonen et al., 2001; Ostrom et al., 2000; Selkirk et al., 2001; Shreeve et al., 2000; Theroux et al., 1996; Zaworski et al., 1999). However, it is likely that similar variations in the relative expression of the partners of the signalling cascade occur in tissues, and this adds further complexity to the cellular responses to a same receptor that depends on the tissue/organ where it is examined.
For decades, it has been generally proposed that a given receptor always interacts with a particular GTP-binding protein (G-protein) or with multiple G-proteins within one family. However, for several G-protein-coupled receptors (GPCR), it now becomes generally accepted that simultaneous functional coupling with distinct unrelated G-proteins can be observed, leading to the activation of multiple intracellular effectors with distinct efficacies and/or potencies. Multiplicity in G-protein coupling is frequently observed in artificial expression systems where high densities of receptors are obtained, raising the question of whether such complex signalling reveals artefactual promiscuous coupling or is a genuine property of GPCRs. Multiple biochemical and pharmacological evidence in favour of an intrinsic property of GPCRs were obtained in recent studies. Thus, there are now many examples showing that the coupling to multiple signalling pathways is dependent on the agonist used (agonist trafficking of receptor signals). In addition, the different couplings were demonstrated to involve distinct molecular determinants of the receptor and to show distinct desensitisation kinetics. Such multiplicity of signalling at the level of G-protein coupling leads to a further complexity in the functional response to agonist stimulation of one of the most elaborate cellular transmission systems. Indeed, the physiological relevance of such versatility in signalling associated with a single receptor requires the existence of critical mechanisms of dynamic regulation of the expression, the compartmentalisation, and the activity of the signalling partners. This review aims at summarising the different studies that support the concept of multiplicity of G-protein coupling. The physiological and pharmacological relevance of this coupling promiscuity will be discussed.
The role of receptor structure in determining adenosine receptor activity
2000, Pharmacology and Therapeutics
Adenosine produces a wide variety of physiological effects through the activation of cell surface adenosine receptors (ARs). ARs are members of the G-protein-coupled receptor family, and currently, four subtypes, the A₁AR, A_2AAR, A_2BAR, and A₃AR, are recognized. This review focuses on the role of receptor structure in governing various facets of AR activity. Ligand-binding properties of ARs are primarily dictated by amino acids in the transmembrane domains of the receptors, although a role for extracellular domains of certain ARs has been suggested. Studies have identified certain amino acids conserved amongst AR subtypes that are critical for ligand recognition, as well as additional residues that may differentiate between agonist and antagonist ligands. Receptor regions responsible for activation of G_s have been identified for the A_2AAR. The location of these intracellular sites is consistent with findings described for other G-protein-coupled receptors. Site-directed mutagenesis has been employed to analyze the structural basis for the differences in the kinetics of the desensitization response displayed by various AR subtypes. For the A_2AAR and A₃AR, agonist-stimulated phosphorylation of the AR, presumably via a G-protein receptor kinase, has been shown to occur. For these AR subtypes, intracellular regions or individual amino acids that may be targets for this phosphorylation have been identified. Finally, the role of A₁AR gene structure in regulating the expression of this AR subtype is reviewed.
α<inf>1</inf>-adrenergic receptor subtype mRNAs are differentially regulated by α<inf>1</inf>-Adrenergic and other hypertrophic stimuli in cardiac myocytes in culture and in vivo: Repression of α<inf>1B</inf> and α<inf>1D</inf> but induction of α<inf>1C</inf>
1996, Journal of Biological Chemistry
The three cloned α₁-adrenergic receptor (AR) subtypes, α^1B, α^1C, and α^1D, can all couple to the same effector, phospholipase C, and the reason(s) for conservation of multiple subtypes remain uncertain. All three α₁-ARs are expressed natively in cultured neonatal rat cardiac myocytes, where chronic exposure to the agonist catecholamine norepinephrine (NE) induces hypertrophic growth and gene transcription. We show here, using RNase protection, that the α₁-AR subtype mRNAs respond in distinctly different ways during prolonged NE exposure (12-72 h). α^1B and α^1D mRNA levels were repressed by NE, whereas α^1C mRNA was induced. Changes in mRNA levels were mediated by an α₁-AR, were not explained by altered mRNA stability, and were reflected in receptor proteins by [³H]prazosin binding. α₁-AR-stimulated phosphoinositide hydrolysis and myocyte growth were not desensitized. Three other hypertrophic agonists in culture, endothelin-1, PGF2α, and phorbol 12-myristate 13-acetate, also induced α^1C mRNA and repressed α^1B mRNA. In myocytes from hearts with pressure overload hypertrophy, α₁ mRNA changes were identical to those produced by NE in culture. These results provide the first example of a difference in regulation among α₁-AR subtypes expressed natively in the same cell. Transcriptional induction of the α^1C-AR could be a mechanism for sustained growth signaling through this receptor and is a common feature of a hypertrophic phenotype in cardiac myocytes.
α<inf>1</inf>-Adrenergic Receptor Subtypes and Signal Transduction
1996, Pharmacochemistry Library
Two pharmacologically distinct α₁-adrenergic receptors (α_1A and α_1B) have been described in animal and human tissues using selective antagonists and site-directed alkylating agents, whereas three different α₁-adrenergic receptor subtypes have been cloned (α_1b, α_1c, and α_1a/d), all of which are members of the G protein linked receptor superfamily. The relationship between native and cloned subtypes was a source of much confusion. However, recently a consensus was reached that the α_1c clone encodes the pharmacological α_1A subtype, and that both the α_1b and α_1a/d clones encode receptors with α_1B-like pharmacology. It is now agreed that the cloned and native receptors should be called α_1a/α_1A, α_1b/α_1B, and α_1d/α_1D, and the α_1c designation has been dropped.
Initial studies in isolated smooth muscle tissue suggested that the pharmacologically defined α_1A subtype preferentially activates voltage-gated Ca²⁺ influx while the α_1B subtype stimulates formation of inositol (1,4,5) trisphosphate (IP₃*) and mobilizes intracellular Ca²⁺ stores. Although these two signalling pathways are generally associated with different receptor subtypes, further work with both native and recombinant subtypes has provided exceptions to this rule. For example, α_1A-adrenergic receptors are linked to inositol phosphate (InsP) formation and mobilization of intracellular Ca²⁺ in human SK-N-MC neuroblastoma cells, and α_1b- and α_1d-adrenergic receptors are linked to voltage-gated Ca²⁺ influx in rat medullary thyroid carcinoma (rMTC 6-23) cells. The exact mechanisms by which α₁-adrenergic receptor subtypes initiate these signals in native cells and tissues are still unclear.
When expressed in mammalian cells, all three cloned subtypes can stimulate InsP formation and release intracellular Ca²⁺, however the efficiency with which each subtype couples to this signalling system appears to vary (α_1a > α_1b > α_1d). No model system is currently suitable for similar studies examining α₁-adrenergic receptor modulation of voltagegated Ca²⁺ influx in response to transfection of the cloned subtypes. These subtypes also activate other second messenger systems following transfection, including phospholipase A₂ (PLA₂) and adenylate cyclase. Use of inducible vectors to titrate the density of each cloned subtype in transfected cell lines allows direct determination of the efficacy of each subtype in activating second messenger responses.

View all citing articles on Scopus

View full text

Inducible expression of α1B-adrenoceptors in DDT1 MF-2 cells: comparison of receptor density and response

Abstract

J. Biol. Chem.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

J. Biol. Chem.

Eur. J. Pharmacol.

Molecular cloning and expression of the cDNA for the hamster α1-adrenergic receptor

Control of gene expression in eukaryotic cells using the lac repressor system

Strategies in Mol. Biol.

Comparison of α1-adrenergic receptor subtypes and signal transduction in SK-N-MC and NB41A3 neuronal cell lines

Mol. Pharmacol.

α1-Adrenergic receptor subtypes

Ann. Rev. Pharmacol. Toxicol.

Increased voltage-dependent calcium influx produced by α1B-adrenergic receptor activation in rat medullary thyroid carcinoma 6–23 cells

Mol. Pharmacol.

Inducible expression of α_1B-adrenoceptors in DDT₁ MF-2 cells: comparison of receptor density and response

Molecular cloning and expression of the cDNA for the hamster α₁-adrenergic receptor

Comparison of α₁-adrenergic receptor subtypes and signal transduction in SK-N-MC and NB41A3 neuronal cell lines

α₁-Adrenergic receptor subtypes

Increased voltage-dependent calcium influx produced by α_1B-adrenergic receptor activation in rat medullary thyroid carcinoma 6–23 cells