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Papers by John Pediani

Confocal images used to generate spatial intensity distribution analysis data for the publication... more Confocal images used to generate spatial intensity distribution analysis data for the publication figures 1 B and 4 A + C

Confocal images used to generate spatial intensity distribution analysis data for the publication... more Confocal images used to generate spatial intensity distribution analysis data for the publication figure 5 B

Confocal images used to generate spatial intensity distribution analysis data for the publication... more Confocal images used to generate spatial intensity distribution analysis data for the publication figure 3 F

Confocal images used to generate spatial intensity distribution analysis data for the publication... more Confocal images used to generate spatial intensity distribution analysis data for the publication figure 3 A

The FEBS journal, Jan 26, 2018

Activation of the Histamine-3 receptor (H3R) is involved in memory processes and cognitive action... more Activation of the Histamine-3 receptor (H3R) is involved in memory processes and cognitive action, whilst blocking H3R activation can slow the progression of neurological disorders such as Alzheimer's disease, schizophrenia and narcolepsy. To date however no direct way to examine the activation of H3R has been utilised. Here, we describe a novel biosensor which can visualise the activation of H3R through an intramolecular Fluorescence Resonance Energy Transfer (FRET) signal. To achieve this, we constructed an intramolecular H3R FRET sensor with cyan fluorescent protein (CFP) attached at the C-terminus and yellow fluorescent protein (YFP) inserted into the third intracellular loop. The sensor was found to internalize normally on agonist treatment. We measured FRET signals between the donor CFP and the acceptor YFP in living cells in real time, the results of which indicated that H3R agonist treatment (imetit or histamine) increases the FRET signal in a time- and concentration-dep...

American journal of physiology. Cell physiology, 2017

Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a re... more Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 μM)], inhibit mitochondrial function [sodium azide (75 μM), rotenone (1 μM), ...

Scientific reports, Jan 18, 2017

The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recent... more The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D3R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D3R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D3R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They show...

The Biochemical journal, May 19, 2017

Previous studies have indicated that the G protein-coupled secretin receptor is present as a homo... more Previous studies have indicated that the G protein-coupled secretin receptor is present as a homo-dimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated, is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and Spatial Intensity Distribution Analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed demonstration that the Epidermal Growth Factor receptor is predominantly monomeric in the absence of ligand and whilst wild type receptor was rapidly converted to a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that at moderate expression levels the secretin receptor exists as a mixture of monomeric a...

The Journal of biological chemistry, Jan 14, 2016

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently... more Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands is unknown. Herein we address these topics for the M1muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by employing Spatial Intensity Distribution Analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules.microm(-2)of the human muscarinic M1receptor identified an ~75/25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter-term treatment with the selective M1antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepin...

Journal of Anatomy

There is no published description of the distribution of free Ca2+, nor of the distribution of Ca... more There is no published description of the distribution of free Ca2+, nor of the distribution of Ca(2+)-ATPase activity associated with the maintenance of low axoplasmic Ca2+ concentrations, in normal central myelinated nerve fibres. We have used the oxalate-pyroantimonate technique to localise free Ca2+, together with the lead-citrate technique to localise Ca(2+)-ATPase activity within myelinated fibres from the adult guinea-pig optic nerve. Pyroantimonate precipitate occurred within the axoplasm at nodes of Ranvier and the internode, at areas of myelin disruption, within Schmidt-Lanterman incisures (SLI) and glial paranodal loops. But precipitate was absent from the axoplasm beneath SLI and at the paranode. Ca(2+)-ATPase activity was localised in axonal smooth endoplasmic reticulum (SER), the outer membrane of mitochondria, the nodal axolemma, the glial membranes of the paranodal loops, the SLI and the external aspect of the myelin sheath. We have demonstrated large domains within t...

The Journal of biological chemistry, Jan 30, 2015

The questions of whether G proteincoupled receptors exist as monomers, dimers and/or oligomers an... more The questions of whether G proteincoupled receptors exist as monomers, dimers and/or oligomers and if these species interconvert in a ligand-dependent manner are amongst the most contentious current issues in biology. When employing Spatial Intensity Distribution Analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membraneassociated form of monomeric eGFP or of a tandem of this fluorophore, the eGFP-tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the Epidermal Growth Factor receptor demonstrated that although largely a monomer in the basal state this receptor rapidly became predominantly dimeric upon addition of its ligand Epidermal Growth Factor. In cells induced to express an eGFPtagged form of the serotonin 5-HT2C receptor global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed Spatial Intensity Distribu...

The Journal of pharmacology and experimental therapeutics, 2000

Cellular distribution and binding characteristics of native alpha(1)-adrenoceptors (ARs) were det... more Cellular distribution and binding characteristics of native alpha(1)-adrenoceptors (ARs) were determined in a live, single, human smooth muscle cell (SMC) with confocal laser scanning microscopy and a fluorescent ligand, BODIPY-FL prazosin (QAPB). This allowed single-cell competitive ligand binding and showed that 40% of alpha(1)-AR-binding sites in native cells are intracellular. QAPB had high affinity and acted as a nonselective, competitive antagonist versus [(3)H]prazosin at cloned human alpha(1a)-, alpha(1b)-, and alpha(1d)-AR subtypes on membrane preparations and whole cells. RS100329 had 70-fold selectivity for alpha(1a)-ARs versus alpha(1b)- and alpha(1d)-ARs, validating its use to identify this subtype. In similar cells QAPB-associated fluorescence provided quantitative data analogous and comparable to [(3)H]prazosin binding in whole cells. In human, dissociated, prostatic smooth muscle cells QAPB-associated fluorescence binding exhibited specific high-affinity binding prop...

The Journal of pharmacology and experimental therapeutics, 2000

Phe-activated Ca(2+) signals recorded from single rat-1 fibroblasts stably expressing the bovine ... more Phe-activated Ca(2+) signals recorded from single rat-1 fibroblasts stably expressing the bovine alpha(1a)-adrenoceptor (AR) were characterized and used to analyze functional agonist-antagonist interactions. The response to Phe was initiated by the mobilization of stored Ca(2+) and subsequently sustained by receptor-regulated Ca(2+) influx. The selective alpha(1A)-AR agonist (R)-A-61603 was 141-fold more potent as an agonist than Phe. This potency ratio was consistent with the pharmacology of the native alpha(1A)-ARs. Functional responses evoked by concentrations of Phe of more than 0. 3 microM displayed fade, which could be explained by agonist-dependent depletion of Ca(2+) stores. The antagonists tested did not conform to the predictions of the Schild equation for competitive antagonism as expected from the nonequilibrium nature of the response. The antagonist potency series WB4101 > or = prazosin > BMY7378, however, was consistent with alpha(1A)-ARs. Antagonism exhibited by...

Molecular Pharmacology, 2005

When expressed via an inducible promoter in human embryonic kidney 293 cells, the rat Mas-related... more When expressed via an inducible promoter in human embryonic kidney 293 cells, the rat Mas-related gene D (rMrgD) receptor responded to ␤-alanine but not L-alanine by elevating intracellular [Ca 2ϩ ], stimulating phosphorylation of the mitogenactivated protein kinases known as extracellular signal-regulated kinase (ERK) 1 and ERK2 and translocating from the plasma membrane to punctate intracellular vesicles. By contrast, the related rat Mas-related gene E (rMrgE) receptor did not respond to ␤-alanine. Coexpression of rMrgD with rMrgE, which occurs in peripheral nociceptive neurons, allowed coimmunoprecipitation of the two receptors and resulted in the detection of cell surface rMrgD-rMrgE heterodimers via timeresolved fluorescence resonance energy transfer. These interactions increased the potency of ␤-alanine to phosphorylate Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

Molecular Pharmacology, 2004

Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence reson... more Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence resonance energy transfer and the functional complementation of pairs of inactive receptor-G protein fusion proteins, the human ␣ 1A-1-adrenoceptor was shown to form homodimeric/oligomeric complexes when expressed in human embryonic kidney (HEK) 293 cells. Saturation bioluminescence resonance energy transfer studies indicated the ␣ 1A-1-adrenoceptor homodimer interactions to be high affinity and some 75 times greater than interactions between the ␣ 1A-1-adrenoceptor and the ␦ opioid peptide receptor. Only a fraction of the ␣ 1A-1-adrenoceptors was at the plasma membrane of HEK293 cells at steady state. However, dimers of ␣ 1A-1-adrenoceptors were also present in intracellular membranes, and the dimer status of those delivered to the cell surface was unaffected by the presence of agonist. Splice variation can generate at least three forms of the human ␣ 1A-1-adrenoceptor with differences limited to the C-terminal tail. Each of the ␣ 1A-1 , ␣ 1A-2a , and ␣ 1A-3a-adrenoceptor splice variants formed homodimers/oligomers, and all combinations of these splice variants were able to generate heterodimeric/oligomeric interactions. Despite the coexpression of these splice variants in human tissues that possess the pharmacologically defined ␣ 1L-adrenoceptor binding site, coexpression of any pair in HEK293 cells failed to generate ligand binding characteristic of the ␣ 1L-adrenoceptor.

Journal of Biological Chemistry, 2002

Journal of Biological Chemistry, 2001

Transfection of either the ␣ 1b-adrenoreceptor or G␣ 11 into a fibroblast cell line derived from ... more Transfection of either the ␣ 1b-adrenoreceptor or G␣ 11 into a fibroblast cell line derived from a G␣ q /G␣ 11 double knockout mouse failed to produce elevation of intracellular [Ca 2؉ ] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the ␣ 1b-adrenoreceptor with the palmitoylation-resistant C9S,C10S G␣ 11 also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S G␣ 11 or C10S G␣ 11. Expression of a fusion protein between the ␣ 1b-adrenoreceptor and G␣ 11 allowed [Ca 2؉ ] i elevation, and this was also true for a fusion protein between the ␣ 1b-adrenoreceptor and C9S,C10S G␣ 11 , since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin ␣, as a ␤⅐␥-sequestering agent, fully attenuated the Ca 2؉ signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type G␣ 11 were also targets for agonist-regulated [ 3 H]palmitoylation and bound [ 35 S]guanosine 5-3-O-(thio)triphosphate (GTP␥S) in an agonist concentration-dependent manner. The potency of agonist to stimulate [ 35 S]GTP␥S binding was unaffected by the palmitoylation potential of either receptor or G protein. These studies provide clear evidence for coordinated, agonist-mediated regulation of the post-translational acylation of both a receptor and partner G protein and demonstrate the capacity of such fusions to bind and then release ␤⅐␥ complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca 2؉ signaling in EF88 cells by such fusion proteins is mediated via release of the G protein ␤⅐␥ complex.

Confocal images used to generate spatial intensity distribution analysis data for the publication... more Confocal images used to generate spatial intensity distribution analysis data for the publication figures 1 B and 4 A + C

Confocal images used to generate spatial intensity distribution analysis data for the publication... more Confocal images used to generate spatial intensity distribution analysis data for the publication figure 5 B

Confocal images used to generate spatial intensity distribution analysis data for the publication... more Confocal images used to generate spatial intensity distribution analysis data for the publication figure 3 F

Confocal images used to generate spatial intensity distribution analysis data for the publication... more Confocal images used to generate spatial intensity distribution analysis data for the publication figure 3 A

The FEBS journal, Jan 26, 2018

Activation of the Histamine-3 receptor (H3R) is involved in memory processes and cognitive action... more Activation of the Histamine-3 receptor (H3R) is involved in memory processes and cognitive action, whilst blocking H3R activation can slow the progression of neurological disorders such as Alzheimer's disease, schizophrenia and narcolepsy. To date however no direct way to examine the activation of H3R has been utilised. Here, we describe a novel biosensor which can visualise the activation of H3R through an intramolecular Fluorescence Resonance Energy Transfer (FRET) signal. To achieve this, we constructed an intramolecular H3R FRET sensor with cyan fluorescent protein (CFP) attached at the C-terminus and yellow fluorescent protein (YFP) inserted into the third intracellular loop. The sensor was found to internalize normally on agonist treatment. We measured FRET signals between the donor CFP and the acceptor YFP in living cells in real time, the results of which indicated that H3R agonist treatment (imetit or histamine) increases the FRET signal in a time- and concentration-dep...

American journal of physiology. Cell physiology, 2017

Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a re... more Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 μM)], inhibit mitochondrial function [sodium azide (75 μM), rotenone (1 μM), ...

Scientific reports, Jan 18, 2017

The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recent... more The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D3R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D3R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D3R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They show...

The Biochemical journal, May 19, 2017

Previous studies have indicated that the G protein-coupled secretin receptor is present as a homo... more Previous studies have indicated that the G protein-coupled secretin receptor is present as a homo-dimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated, is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and Spatial Intensity Distribution Analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed demonstration that the Epidermal Growth Factor receptor is predominantly monomeric in the absence of ligand and whilst wild type receptor was rapidly converted to a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that at moderate expression levels the secretin receptor exists as a mixture of monomeric a...

The Journal of biological chemistry, Jan 14, 2016

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently... more Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands is unknown. Herein we address these topics for the M1muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by employing Spatial Intensity Distribution Analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules.microm(-2)of the human muscarinic M1receptor identified an ~75/25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter-term treatment with the selective M1antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepin...

Journal of Anatomy

There is no published description of the distribution of free Ca2+, nor of the distribution of Ca... more There is no published description of the distribution of free Ca2+, nor of the distribution of Ca(2+)-ATPase activity associated with the maintenance of low axoplasmic Ca2+ concentrations, in normal central myelinated nerve fibres. We have used the oxalate-pyroantimonate technique to localise free Ca2+, together with the lead-citrate technique to localise Ca(2+)-ATPase activity within myelinated fibres from the adult guinea-pig optic nerve. Pyroantimonate precipitate occurred within the axoplasm at nodes of Ranvier and the internode, at areas of myelin disruption, within Schmidt-Lanterman incisures (SLI) and glial paranodal loops. But precipitate was absent from the axoplasm beneath SLI and at the paranode. Ca(2+)-ATPase activity was localised in axonal smooth endoplasmic reticulum (SER), the outer membrane of mitochondria, the nodal axolemma, the glial membranes of the paranodal loops, the SLI and the external aspect of the myelin sheath. We have demonstrated large domains within t...

The Journal of biological chemistry, Jan 30, 2015

The questions of whether G proteincoupled receptors exist as monomers, dimers and/or oligomers an... more The questions of whether G proteincoupled receptors exist as monomers, dimers and/or oligomers and if these species interconvert in a ligand-dependent manner are amongst the most contentious current issues in biology. When employing Spatial Intensity Distribution Analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membraneassociated form of monomeric eGFP or of a tandem of this fluorophore, the eGFP-tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the Epidermal Growth Factor receptor demonstrated that although largely a monomer in the basal state this receptor rapidly became predominantly dimeric upon addition of its ligand Epidermal Growth Factor. In cells induced to express an eGFPtagged form of the serotonin 5-HT2C receptor global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed Spatial Intensity Distribu...

The Journal of pharmacology and experimental therapeutics, 2000

Cellular distribution and binding characteristics of native alpha(1)-adrenoceptors (ARs) were det... more Cellular distribution and binding characteristics of native alpha(1)-adrenoceptors (ARs) were determined in a live, single, human smooth muscle cell (SMC) with confocal laser scanning microscopy and a fluorescent ligand, BODIPY-FL prazosin (QAPB). This allowed single-cell competitive ligand binding and showed that 40% of alpha(1)-AR-binding sites in native cells are intracellular. QAPB had high affinity and acted as a nonselective, competitive antagonist versus [(3)H]prazosin at cloned human alpha(1a)-, alpha(1b)-, and alpha(1d)-AR subtypes on membrane preparations and whole cells. RS100329 had 70-fold selectivity for alpha(1a)-ARs versus alpha(1b)- and alpha(1d)-ARs, validating its use to identify this subtype. In similar cells QAPB-associated fluorescence provided quantitative data analogous and comparable to [(3)H]prazosin binding in whole cells. In human, dissociated, prostatic smooth muscle cells QAPB-associated fluorescence binding exhibited specific high-affinity binding prop...

The Journal of pharmacology and experimental therapeutics, 2000

Phe-activated Ca(2+) signals recorded from single rat-1 fibroblasts stably expressing the bovine ... more Phe-activated Ca(2+) signals recorded from single rat-1 fibroblasts stably expressing the bovine alpha(1a)-adrenoceptor (AR) were characterized and used to analyze functional agonist-antagonist interactions. The response to Phe was initiated by the mobilization of stored Ca(2+) and subsequently sustained by receptor-regulated Ca(2+) influx. The selective alpha(1A)-AR agonist (R)-A-61603 was 141-fold more potent as an agonist than Phe. This potency ratio was consistent with the pharmacology of the native alpha(1A)-ARs. Functional responses evoked by concentrations of Phe of more than 0. 3 microM displayed fade, which could be explained by agonist-dependent depletion of Ca(2+) stores. The antagonists tested did not conform to the predictions of the Schild equation for competitive antagonism as expected from the nonequilibrium nature of the response. The antagonist potency series WB4101 > or = prazosin > BMY7378, however, was consistent with alpha(1A)-ARs. Antagonism exhibited by...

Molecular Pharmacology, 2005

When expressed via an inducible promoter in human embryonic kidney 293 cells, the rat Mas-related... more When expressed via an inducible promoter in human embryonic kidney 293 cells, the rat Mas-related gene D (rMrgD) receptor responded to ␤-alanine but not L-alanine by elevating intracellular [Ca 2ϩ ], stimulating phosphorylation of the mitogenactivated protein kinases known as extracellular signal-regulated kinase (ERK) 1 and ERK2 and translocating from the plasma membrane to punctate intracellular vesicles. By contrast, the related rat Mas-related gene E (rMrgE) receptor did not respond to ␤-alanine. Coexpression of rMrgD with rMrgE, which occurs in peripheral nociceptive neurons, allowed coimmunoprecipitation of the two receptors and resulted in the detection of cell surface rMrgD-rMrgE heterodimers via timeresolved fluorescence resonance energy transfer. These interactions increased the potency of ␤-alanine to phosphorylate Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

Molecular Pharmacology, 2004

Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence reson... more Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence resonance energy transfer and the functional complementation of pairs of inactive receptor-G protein fusion proteins, the human ␣ 1A-1-adrenoceptor was shown to form homodimeric/oligomeric complexes when expressed in human embryonic kidney (HEK) 293 cells. Saturation bioluminescence resonance energy transfer studies indicated the ␣ 1A-1-adrenoceptor homodimer interactions to be high affinity and some 75 times greater than interactions between the ␣ 1A-1-adrenoceptor and the ␦ opioid peptide receptor. Only a fraction of the ␣ 1A-1-adrenoceptors was at the plasma membrane of HEK293 cells at steady state. However, dimers of ␣ 1A-1-adrenoceptors were also present in intracellular membranes, and the dimer status of those delivered to the cell surface was unaffected by the presence of agonist. Splice variation can generate at least three forms of the human ␣ 1A-1-adrenoceptor with differences limited to the C-terminal tail. Each of the ␣ 1A-1 , ␣ 1A-2a , and ␣ 1A-3a-adrenoceptor splice variants formed homodimers/oligomers, and all combinations of these splice variants were able to generate heterodimeric/oligomeric interactions. Despite the coexpression of these splice variants in human tissues that possess the pharmacologically defined ␣ 1L-adrenoceptor binding site, coexpression of any pair in HEK293 cells failed to generate ligand binding characteristic of the ␣ 1L-adrenoceptor.

Journal of Biological Chemistry, 2002

Journal of Biological Chemistry, 2001

Transfection of either the ␣ 1b-adrenoreceptor or G␣ 11 into a fibroblast cell line derived from ... more Transfection of either the ␣ 1b-adrenoreceptor or G␣ 11 into a fibroblast cell line derived from a G␣ q /G␣ 11 double knockout mouse failed to produce elevation of intracellular [Ca 2؉ ] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the ␣ 1b-adrenoreceptor with the palmitoylation-resistant C9S,C10S G␣ 11 also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S G␣ 11 or C10S G␣ 11. Expression of a fusion protein between the ␣ 1b-adrenoreceptor and G␣ 11 allowed [Ca 2؉ ] i elevation, and this was also true for a fusion protein between the ␣ 1b-adrenoreceptor and C9S,C10S G␣ 11 , since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin ␣, as a ␤⅐␥-sequestering agent, fully attenuated the Ca 2؉ signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type G␣ 11 were also targets for agonist-regulated [ 3 H]palmitoylation and bound [ 35 S]guanosine 5-3-O-(thio)triphosphate (GTP␥S) in an agonist concentration-dependent manner. The potency of agonist to stimulate [ 35 S]GTP␥S binding was unaffected by the palmitoylation potential of either receptor or G protein. These studies provide clear evidence for coordinated, agonist-mediated regulation of the post-translational acylation of both a receptor and partner G protein and demonstrate the capacity of such fusions to bind and then release ␤⅐␥ complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca 2؉ signaling in EF88 cells by such fusion proteins is mediated via release of the G protein ␤⅐␥ complex.