Biomedical subjects

Kenneth P Minneman

Publications and source records attributed to Kenneth P Minneman.

At least 19 recordsLinked to original sources

PubMed2006

New pyrimido[5,4-b]indoles and [1]benzothieno[3,2-d]pyrimidines: high affinity ligands for the alpha(1)-adrenoceptor subtypes.

A number of new pyrimido[5,4-b]indole and [1]benzothieno[3,2-d]pyrimidine derivatives were synthesized and evaluated for their binding and functional properties at alpha(1)-adrenergic receptor (alpha(1)-AR) subtypes. They behaved as potent alpha(1)-AR antagonists. In binding experiments, some of them (RC24 and RC23) showed very high affinity for the alpha(1D)-AR subtype.

Animals↗

PubMed2006

Heterodimerization and surface localization of G protein coupled receptors.

G protein coupled receptors (GPCRs) are one of the largest human gene families, and are targets for many important therapeutic drugs. Over the last few years, there has been a major paradigm shift in our understanding of how these receptors function. Formerly, GPCRs were thought to exist as monomers that, upon agonist occupation, activated a heterotrimeric G protein to alter the concentrations of specific second messengers. Until recently, this relatively linear cascade has been the standard paradigm for signaling by these molecules. However, it is now clear that this model is not adequate to explain many aspects of GPCR function. We now know that many, if not most, GPCRs form homo- and/or hetero-oligomeric complexes and interact directly with intracellular proteins in addition to G proteins. It now appears that many GPCRs may not function independently, but might more accurately be described as subunits of large multi-protein signaling complexes. These observations raise many important new questions; some of which include: (1) how many functionally and pharmacologically distinct receptor subtypes exist in vivo? (2) Which GPCRs physically associate, and in what stochiometries? (3) What are the roles of individual subunits in binding ligand and activating responses? (4) Are the pharmacological or signaling properties of GPCR heterodimers different from monomers? Since these receptors are the targets for a large number of clinically useful compounds, such information is likely to be of direct therapeutic importance, both in understanding how existing drugs work, but also in discovering novel compounds to treat disease.

Dimerization↗

PubMed2006

New 1,2,3,9-tetrahydro-4H-carbazol-4-one derivatives: analogues of HEAT as ligands for the alpha1-adrenergic receptor subtypes.

With the aim to develop new ligands able to discriminate among the three subtypes of alpha1-adrenergic receptors (alpha1A-AR, alpha1B-AR, and alpha1D-AR), a series of new 1,2,3,9-tetrahydro-4H-carbazol-4-ones bearing a 3-[[[2-(4-hydroxyphenyl)ethyl]amino]methyl] or a 3-[[4-(2-substitutedphenyl)piperazin-1-yl]methyl] side chain were synthesized. The general structure of the new compounds is reminiscent of HEAT and RN5, two potent alpha1-AR antagonists which show high affinities for all three alpha1-AR subtypes. Some derivatives in which one ring of the tetrahydrocarbazolone system was opened were also prepared. Compounds were tested in radioligand binding assays on human cloned alpha1A-AR, alpha1B-AR, and alpha1D-AR subtypes stably expressed in HEK293 cells. They showed moderate to good affinities, although their selectivity among the receptor subtypes hardly reached one order of magnitude.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed2006

Syntrophins regulate alpha1D-adrenergic receptors through a PDZ domain-mediated interaction.

To find novel cytoplasmic binding partners of the alpha1D-adrenergic receptor (AR), a yeast two-hybrid screen using the alpha1D-AR C terminus as bait was performed on a human brain cDNA library. Alpha-syntrophin, a protein containing one PDZ domain and two pleckstrin homology domains, was isolated in this screen as an alpha1D-AR-interacting protein. Alpha-syntrophin specifically recognized the C terminus of alpha1D- but not alpha1A- or alpha1B-ARs. In blot overlay assays, the PDZ domains of syntrophin isoforms alpha, beta1, and beta2 but not gamma1 or gamma2 showed strong selective interactions with the alpha1D-AR C-tail fusion protein. In transfected human embryonic kidney 293 cells, full-length alpha1D- but not alpha1A- or alpha1B-ARs co-immunoprecipitated with syntrophins, and the importance of the receptor C terminus for the alpha1D-AR/syntrophin interaction was confirmed using chimeric receptors. Mutation of the PDZ-interacting motif at the alpha1D-AR C terminus markedly decreased inositol phosphate formation stimulated by norepinephrine but not carbachol in transfected HEK293 cells. This mutation also dramatically decreased alpha1D-AR binding and protein expression. In addition, stable overexpression of alpha-syntrophin significantly increased alpha1D-AR protein expression and binding but did not affect those with a mutated PDZ-interacting motif, suggesting that syntrophin plays an important role in maintaining receptor stability by directly interacting with the receptor PDZ-interacting motif. This direct interaction may provide new information about the regulation of alpha1D-AR signaling and the role of syntrophins in modulating G protein-coupled receptor function.

Animals↗

PubMed2005

Heterodimers of alpha1B- and alpha1D-adrenergic receptors form a single functional entity.

Heterologous expression of alpha(1D)-adrenergic receptors (alpha(1D)-ARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with alpha(1B)-ARs promotes surface localization of alpha(1D)-ARs. Here, we report that the alpha(1B)-/alpha(1D)-AR interaction has significant effects on the pharmacology and signaling of the receptors, in addition to the effects on trafficking described previously. Upon coexpression of alpha(1B)-ARs and epitope-tagged alpha(1D)-ARs in both human embryonic kidney 293 and DDT(1)MF-2 cells, alpha(1D)-AR binding sites were not detectable with the alpha(1D)-AR selective antagonist 8-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione (BMY 7378), despite the ability to detect alpha(1D)-AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the alpha(1B)-AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in alpha(1B)/alpha(1D)-AR-expressing cells, revealing that alpha(1D)-ARs were expressed but did not bind BMY 7378 with high affinity. Studies of norepinephrine-stimulated inositol phosphate formation showed that maximal responses were greatest in alpha(1B)/alpha(1D)-AR-coexpressing cells. Stable coexpression of an uncoupled mutant alpha(1B)-AR (Delta12) with alpha(1D)-ARs resulted in increased responses to norepinephrine. However, Schild plots for inhibition of norepinephrine-stimulated inositol phosphate formation showed a single low-affinity site for BMY 7378. Thus, our findings suggest that alpha(1B)/alpha(1D)-AR heterodimers form a single functional entity with enhanced functional activity relative to either subtype alone and a novel pharmacological profile. These data may help to explain why alpha(1D)-ARs are often pharmacologically undetectable in native tissues when they are coexpressed with alpha(1B)-ARs.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed2005

Selective inhibition of alpha1A-adrenergic receptor signaling by RGS2 association with the receptor third intracellular loop.

Regulators of G-protein signaling (RGS) proteins act directly on Galpha 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 alpha1A-adrenergic receptor (AR) in vitro, and is recruited by the unstimulated alpha1A-AR to the plasma membrane in cells to inhibit receptor and Gq/11 signaling. This interaction was specific, because RGS2 did not interact with the highly homologous alpha1B- or alpha1D-ARs, and the closely related RGS16 did not interact with any alpha1-ARs. The N terminus of RGS2 was required for association with alpha1A-ARs and inhibition of signaling, and amino acids Lys219, Ser220, and Arg238 within the alpha1A-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.

Adrenergic alpha-Antagonists↗

PubMed2005

Differential antagonism by conotoxin rho-TIA of contractions mediated by distinct alpha1-adrenoceptor subtypes in rat vas deferens, spleen and aorta.

The ability of the conotoxin rho-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa, to antagonize contractions induced by noradrenaline through activation of alpha1A-adrenoceptors in rat vas deferens, alpha1B-adrenoceptors in rat spleen and alpha1D-adrenoceptors in rat aorta, and to inhibit the binding of [125I]HEAT (2-[[beta-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat alpha1-adrenoceptors was investigated. rho-TIA (100 nM to 1 microM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (pA2 approximately 7.2, n=4). This suggests that rho-TIA is a competitive antagonist of alpha1A- and alpha1D-adrenoceptors with no selectivity between these subtypes. Incubation of rho-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that rho-TIA is a non-competitive antagonist at alpha1B-adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of rho-TIA in inhibiting contractions was examined with similar occupancies (approximately 25%) at each subtype. Its potency (pIC50) was 12 times higher in spleen (8.3+/-0.1, n=4) than in vas deferens (7.2+/-0.1, n=4) or aorta (7.2+/-0.1, n=4). In radioligand binding assays, rho-TIA decreased the number of binding sites (B(max)) in membranes from HEK293 cells expressing the rat alpha1B-adrenoceptors without affecting affinity (K(D)). In contrast, in HEK293 cells expressing rat alpha1A- or alpha1D-adrenoceptors, rho-TIA decreased the K(D) without affecting the B(max). It is concluded that rho-TIA will be useful for distinguishing the role of particular alpha1-adrenoceptor subtypes in native tissues.

Adrenergic alpha-1 Receptor Agonists↗

PubMed2005

Recent progress in alpha1-adrenergic receptor research.

Alpha1-Adrenergic receptors (AR) play an important role in the regulation of physiological responses mediated by norepinephrine and epinephrine, particularly in the cardiovascular system. The three cloned alpha1-AR subtypes (alpha1A, alpha1B, and alpha1D) are G protein-coupled receptors that signal through the Gq/11 signaling pathway, each showing distinct pharmacological properties and tissue distributions. However, due to the lack of highly subtype-selective drugs, the functional roles of individual subtypes are still not clear. Development of new subtype-specific drugs will greatly facilitate the identification of the functions of each subtype. Conopeptide rho-TIA has been found to be a new alpha1B-AR selective antagonist with different modes of inhibition at alpha1-AR subtypes. In addition, recent studies using genetically engineered mice have shed some light on alpha1-AR functions in vivo, especially in the cardiovascular system and brain. Several proteins have been shown to interact directly with particular alpha1-AR, and may be important in regulating receptor function. Receptor heterodimerization has been shown to be important for cell surface expression, signaling and internalization. These new observations are likely to help elucidate the functional roles of individual alpha1-AR subtypes.

Adrenergic alpha-Antagonists↗

PubMed2004

Heterodimerization with beta2-adrenergic receptors promotes surface expression and functional activity of alpha1D-adrenergic receptors.

The alpha1D-adrenergic receptor (alpha1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote alpha1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of alpha1D-AR at the cell surface: alpha1B-AR and beta2-AR. Confocal imaging confirmed that coexpression with beta2-AR resulted in translocation of alpha1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that alpha1D-AR and beta2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total alpha1D-AR binding sites upon coexpression with beta2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with beta2-AR significantly enhanced the coupling of alpha1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of beta2-AR with alpha1D-AR also conferred the ability of alpha1D-AR to cointernalize upon beta2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of alpha1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between alpha1- and beta2-ARs that is mediated through heterodimerization and cross-internalization.

Adrenergic beta-Agonists↗

PubMed2004

Olfactory receptor surface expression is driven by association with the beta2-adrenergic receptor.

Olfactory receptors (ORs) comprise more than half of the large class I G protein-coupled receptor (GPCR) superfamily. Although cloned over a decade ago, little is known about their properties because wild-type ORs do not efficiently reach the cell surface following heterologous expression. Receptor-receptor interactions strongly influence surface trafficking of other GPCRs, and we examined whether a similar mechanism might be involved in OR surface expression. Olfactory neurons are known to express beta-adrenergic receptors (ARs), and we found that coexpression with beta(2)-ARs, but not any other AR subtypes, dramatically increased mouse 71 (M71) OR surface expression in human embryonic kidney 293 cells. A persistent physical interaction between M71 ORs and beta(2)-ARs was shown by coimmunoprecipitation and by cointernalization of the two receptors in response to their specific ligands. Also, coexpression of wild-type M71 ORs with beta(2)-ARs resulted in cAMP responses to the M71 ligand acetophenone. Finally, in situ hybridization studies showed extensive colocalization of M71 OR and beta(2)-AR expression in mouse olfactory epithelium. These data demonstrate the successful heterologous surface expression of a functional wild-type OR and reveal that persistent physical association with other GPCRs can control OR surface expression.

Adrenergic beta-2 Receptor Agonists↗

PubMed2004

Subtype-selective noncompetitive or competitive inhibition of human alpha1-adrenergic receptors by rho-TIA.

The 19-amino acid conopeptide (rho-TIA) was shown previously to antagonize noncompetitively alpha(1B)-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human alpha(1)-AR subtypes (alpha(1A), alpha(1B), and alpha(1D)). Radioligand binding assays showed that rho-TIA was 10-fold selective for human alpha(1B)-over alpha(1A)- and alpha(1D)-ARs. As observed with hamster alpha(1B)-ARs, rho-TIA decreased the number of binding sites (B(max)) for human alpha(1B)-ARs without changing affinity (K(D)), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, rho-TIA had opposite effects at human alpha(1A)-ARs and alpha(1D)-ARs, decreasing K(D) without changing B(max), suggesting it acts competitively at these subtypes. rho-TIA reduced maximal NE-stimulated [(3)H]inositol phosphate formation in HEK293 cells expressing human alpha(1B)-ARs but competitively inhibited responses in cells expressing alpha(1A)- or alpha(1D)-ARs. Truncation mutants showed that the amino-terminal domains of alpha(1B)- or alpha(1D)-ARs are not involved in interaction with rho-TIA. Alanine-scanning mutagenesis of rho-TIA showed F18A had an increased selectivity for alpha(1B)-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at alpha(1B)-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus rho-TIA noncompetitively inhibits alpha(1B)-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at alpha(1B)-ARs.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed2004

Cell surface expression of alpha1D-adrenergic receptors is controlled by heterodimerization with alpha1B-adrenergic receptors.

alpha(1)-Adrenergic receptors (ARs) belong to the large Class I G protein-coupled receptor superfamily and comprise three subtypes (alpha(1A), alpha(1B), and alpha(1D)). Previous work with heterologously expressed C-terminal green fluorescent protein (GFP)-tagged alpha(1)-ARs showed that alpha(1A)- and alpha(1B)-ARs localize to the plasma membrane, whereas alpha(1D)-ARs accumulate intracellularly. We recently showed that alpha(1D)- and alpha(1B)-ARs form heterodimers, whereas alpha(1D)- and alpha(1A)-ARs do not. Here, we examined the role of heterodimerization in regulating alpha(1D)-AR localization using both confocal imaging of GFP- or CFP-tagged alpha(1)-ARs and a luminometer-based surface expression assay in HEK293 cells. Co-expression with alpha(1B)-ARs caused alpha(1D)-ARs to quantitatively translocate to the cell surface, but co-expression with alpha(1A)-ARs did not. Truncation of the alpha(1B)-AR extracellular N terminus or intracellular C terminus had no effect on surface expression of alpha(1D)-ARs, suggesting primary involvement of the hydrophobic core. Co-transfection with an uncoupled mutant alpha(1B)-AR (Delta12alpha(1B)) increased both alpha(1D)-AR surface expression and coupling to norepinephrine-stimulated Ca(2+) mobilization. Finally, GFP-tagged alpha(1D)-ARs were not detected on the cell surface when expressed in rat aortic smooth muscle cells that express no endogenous ARs, but were almost exclusively localized on the surface when expressed in DDT(1)MF-2 cells, which express endogenous alpha(1B)-ARs. These studies demonstrate that alpha(1B)/alpha(1D)-AR heterodimerization controls surface expression and functional coupling of alpha(1D)-ARs, the N- and C-terminal domains are not involved in this interaction, and that alpha(1B)-AR G protein coupling is not required. These observations may be relevant to many other Class I G protein-coupled receptors, where the functional consequences of heterodimerization are still poorly understood.

Amino Acid Sequence↗

PubMed2004

The N terminus of the human alpha1D-adrenergic receptor prevents cell surface expression.

We previously reported that truncation of the N-terminal 79 amino acids of alpha(1D)-adrenoceptors (Delta(1-79)alpha(1D)-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in alpha(1D)-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length alpha(1D)-ARs were found primarily in intracellular compartments, whereas Delta(1-79)alpha(1D)-ARs were translocated to the plasma membrane. This resulted in a 3- to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of alpha(1)-ARs containing the body of one subtype and the N terminus of another (alpha(1A)NT-D, alpha(1B)NT-D, alpha(1D)NT-A, and alpha(1D)NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of alpha(1A)-or alpha(1B)-ARs containing the alpha(1D)-N terminus decreased by 86 to 93%, whereas substitution of alpha(1A)- or alpha(1B)-N termini increased alpha(1D)-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged alpha(1D)NT-B-ARs were found only on the cell surface, whereas GFP-tagged alpha(1B)NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged alpha(1D)- and Delta(1-79)alpha(1D)-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express alpha(1D)-ARs. These findings demonstrate that the N-terminal region of alpha(1D)-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.

Binding Sites↗

PubMed2004

Olfactory receptor localization and function: an emerging role for GPCR heterodimerization.

Research on olfaction has been fraught with considerable frustration because none of the hundreds of olfactory receptors make it to the cell surface on their own when expressed in heterologous systems. Recent work indicates that the heterodimerization of olfactory receptors with beta2-adrenergic receptors results in surface expression of these G protein-coupled receptors. Similar conclusions--that heterodimerization is essential for surface expression of olfactory receptors--have been drawn from research in Drosophila utilizing completely different knockout and functional approaches. Together these findings may unlock the solution to a problem that has plagued the molecular study of olfaction since the cloning of the first olfactory G protein-coupled receptor over twelve years ago.

Animals↗

PubMed2003

Alpha(1)-adrenergic receptor subtypes: non-identical triplets with different dancing partners?

Alpha(1)-adrenergic receptors are one of the three subfamilies of G protein coupled receptors activated by epinephrine and norepinephrine to control important functions in many target organs. Three human subtypes (alpha(1A), alpha(1B), alpha(1D)) are derived from separate genes and are highly homologous in their transmembrane domains but not in their amino or carboxyl termini. Recent advances in our understanding of these "non-identical triplets" include development of knockout mice lacking single or multiple subtypes, new insights into subcellular localization and trafficking, identification of allosteric modulators, and increasing evidence for an important role in brain function. Although all three subtypes activate the same G(q/11) signaling pathway, they also appear to interact with different protein binding partners. Recent evidence suggests they may also form dimers, and may initiate independent signals through pathways yet to be clearly elucidated. Thus, this subfamily represents a common phenomenon of a group of similar but non-identical receptor subtypes activated by the same neurotransmitter, whose individual functional roles remain to be clearly established.

Animals↗

PubMed2003

New pyrimido[5,4-b]indoles as ligands for alpha(1)-adrenoceptor subtypes.

A new series of compounds were designed as structural analogues of the alpha(1)-AR ligand RN5 (4), characterized by a tricyclic 5H-pyrimido[5,4-b]indole-(1H,3H)2,4-dione system connected through an alkyl chain to a phenylpiperazine (PP) moiety. These compounds were synthesized and tested in binding assays on human alpha(1A)-AR, alpha(1B)-AR, and alpha(1D)-AR subtypes expressed in HEK293 cells. Several structural modifications were performed on the PP moiety, the tricyclic system, and the connecting alkyl chain. Many of the new molecules showed a preferential affinity for the alpha(1D)-AR subtype. Some compounds, including 39 and 40, displayed substantial alpha(1D)-AR selectivity with respect to alpha(1A)-AR, alpha(1B)-AR, serotonergic 5-HT(1A), 5-HT(1B), 5-HT(2A), and dopaminergic D(1) and D(2) receptors. Two conformationally rigid analogues of 4, useful for studying the architecture of the receptor/ligand complex, were also prepared and tested. A subset of the new compounds was then used to evolve a preliminary pharmacophore model for alpha(1D)-AR antagonists, based on a more generalized model we had developed for alpha(1)-AR antagonists. This new model rationalized the relationships between structural properties and biological data of the pyrimido[5,4-b]indole compounds, as well as other compounds.

Adrenergic alpha-Antagonists↗

PubMed2003

Mutational uncoupling of alpha1A-adrenergic receptors from G proteins also uncouples mitogenic and transcriptional responses in PC12 cells.

Activation of human alpha1A-adrenergic receptors in PC12 cells causes many second messenger, mitogenic, and transcriptional responses. We examined the role of G protein activation in these responses by uncoupling the receptor through deletion of the first three amino acids from the third intracellular loop (Delta208-210). Expression levels of retrovirus-transfected wild-type and Delta(208-210) alpha1A-adrenergic receptors in PC12 cells were similar and showed identical binding affinities for antagonists. However, the potency of the agonist norepinephrine was increased 9-fold by the Delta (208-210) mutation. In PC12 cells expressing the Delta (208-210) construct, calcium and inositol phosphate responses to norepinephrine were essentially abolished. The strong activation of mitogen-activated protein kinase pathways seen upon stimulation of wild-type alpha1A-adrenergic receptors in PC12 cells was abolished by the Delta (208-210) mutation, as was activation of the tyrosine kinase Pyk2. Norepinephrine also activates several transcriptional reporters through alpha1A-adrenergic receptors in PC12 cells, including reporters for activator protein 1, serum response element, cAMP response element, nuclear factor-kappaB, nuclear factor of activated T cells, gamma-interferon-activated sequence, and signal transducer and activator of transcription. All these transcriptional responses were abolished by the Delta (208-210) mutation. Overexpression of Galpha16 did not rescue any of these responses. These data suggest that known second messenger, mitogenic, and transcriptional effects of alpha1A-adrenergic receptors in PC12 cells all require G protein activation.

Animals↗

PubMed2003

N-terminal truncation of human alpha1D-adrenoceptors increases expression of binding sites but not protein.

The role of the N-terminus of human alpha(1D)-adrenoceptors was examined by deleting the first 79 amino acids (Delta(1-79)) and epitope-tagging to facilitate immunoprecipitation and detection. Following transfection into HEK293 cells, 6- to 13-fold increases in the density of specific [125I]BE 2254 binding sites were observed for both tagged and untagged Delta(1-79)alpha(1D)- compared to full-length alpha(1D)-adrenoceptors, while agonist and antagonist affinities remained unchanged. In contrast, immunoprecipitation of tagged receptors showed that full-length alpha(1D)-adrenoceptor protein was at least twice as abundant as Delta(1-79)alpha(1D)-adrenoceptor protein. Photoaffinity labelling with [125I]arylazidoprazosin showed much more intense labelling of tagged Delta(1-79)alpha(1D)- than of full-length alpha(1D)-adrenoceptors. Substantial N-linked glycosylation of tagged Delta(1-79)alpha(1D)-adrenoceptors was observed, although full-length alpha(1D)-adrenoceptors contain two consensus glycosylation sites but are not glycosylated. These results suggest that N-terminal truncation of alpha(1D)-adrenoceptors enhances processing of a binding competent form in HEK293 cells; and show a clear dissociation between abundance of receptor protein and density of receptor binding sites.

Amino Acid Sequence↗