Cooperativity Manifest in the Binding Properties of Purified Cardiac Muscarinic Receptors

Wreggett, Keith A.; Wells, James W.

doi:10.1074/jbc.270.38.22488

Cited by 159 publications

(188 citation statements)

References 51 publications

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“…For example, agonist binding to GPCRs such as cardiac muscarinic acetylcholine receptors is cooperative (21,22). Furthermore, a recent investigation using ␣1b-adrenergic or histamine H1 receptors fused at their C termini with G␣ 11 has shown that G 11 activation occurs upon co-expression of one fusion consisting of wild type G 11 and a receptor defective in G protein activation with a second fusion protein consisting of a wild type receptor and mutant G 11 that cannot release GDP (54).…”

Section: Discussionmentioning

confidence: 99%

“…In one model, agonist binding to one receptor subunit induces a conformational change that is transmitted allosterically to another receptor subunit, which in turn undergoes a conformational change resulting in G protein activation. This mechanism has been suggested by studies of the GABA(B) receptor heterodimers (16 -20), by cooperative agonist binding to receptors such as muscarinic acetylcholine receptors (21,22), and by studies indicating that heterodimerization can alter the signaling, trafficking, or desensitization of opioid, ␤-adrenergic, bradykinin, or angiotensin II receptors (23)(24)(25)(26)(27). In a second model suggested by studies of GABA(B) receptors (16 -20), agonist binding to the R1 subunit induces movement that relieves its inhibitory action on the R2 subunit, which is freed for G protein activation.…”

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confidence: 99%

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Subunits of a Yeast Oligomeric G Protein-coupled Receptor Are Activated Independently by Agonist but Function in Concert to Activate G Protein Heterotrimers

Chinault

Overton²,

Blumer³

2004

Journal of Biological Chemistry

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G protein-coupled receptors (GPCRs) form dimeric or oligomeric complexes in vivo. However, the function of oligomerization in receptor-mediated G protein activation is unclear. Previous studies of the yeast ␣-factor receptor (STE2 gene product) have indicated that oligomerization promotes signaling. Here we have addressed the mechanism by which oligomerization facilitates G protein signaling by examining the ability of ligand binding-and G protein coupling-defective ␣-factor receptors to form complexes in vivo and to correct their signaling defects when co-expressed (trans complementation). Newly and previously identified receptor mutants indicated that ligand binding involves the exofacial end of transmembrane domain (TM) 4, whereas G protein coupling involves ic1, ic3, the C-terminal tail, and the intracellular ends of TM2 and TM3. Mutant receptors bearing substitutions in these domains formed homo-oligomeric or hetero-oligomeric complexes in vivo, as indicated by results of fluorescence resonance energy transfer experiments. Co-expression of ligand binding-and G protein coupling-defective mutant receptors did not significantly improve signaling. In contrast, co-expression of ic1 and ic3 mutations in trans but not in cis significantly increased signaling efficiency. Therefore, we suggest that subunits of the ␣-factor receptor: 1) are activated independently rather than cooperatively by agonist, and 2) function in a concerted fashion to promote G protein activation, possibly by contacting different subunits or regions of the G protein heterotrimer.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Subunits of a Yeast Oligomeric G Protein-coupled Receptor Are Activated Independently by Agonist but Function in Concert to Activate G Protein Heterotrimers

Chinault

Overton²,

Blumer³

2004

Journal of Biological Chemistry

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“…A distinct and more pronounced heterogeneity has been observed more recently in the binding of antagonists to the M 2 muscarinic receptor and the D 2 dopamine receptor (Wreggett and Wells 1995;Armstrong and Strange 2001;Park et al 2002). In each case, a comparison of two radiolabeled antagonists revealed a difference in apparent capacity such that some receptors appeared to be of anomalously low affinity for one of the ligands.…”

mentioning

confidence: 99%

“…The analysis of cooperative effects in terms of explicit mechanistic models has suggested that the M 2 muscarinic receptor is at least tetrameric (Wreggett and Wells 1995;Chidiac et al 1997;Park et al 2002). Direct biochemical evidence for oligomers derives primarily from measurements of coimmunoprecipitation and energy transfer, which generally have not allowed dimers to be distinguished from larger aggregates.…”

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confidence: 99%

Oligomeric potential of the M₂ muscarinic cholinergic receptor

Park

Wells

2004

Journal of Neurochemistry

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G protein-coupled receptors are known to exist as oligomers. Although such aggregates often are referred to as dimers, there is little direct evidence regarding their oligomeric size. In the present investigation, c-Myc-, FLAG-, and influenza hemagglutinin (HA)-tagged forms of the M 2 muscarinic receptor have been coexpressed in Sf9 cells to probe for aggregates larger than a dimer. Immunochromatography, immunoprecipitation, and immunoblotting were carried out with various combinations of antibodies directed against the different epitopes to demonstrate that all three tagged forms of the receptor can be immunopurified within a single complex. Extracts of the M 2 muscarinic receptor from Sf9 cells therefore contain aggregates that are at least trimeric, and the levels detected point to the existence of larger complexes. The data also suggest that the oligomers coexist with a sizeable population of monomers.

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“…Further details regarding the analyses and related statistical procedures have been described elsewhere [11][12][13][14].…”

Section: Binding Assays and Analysis Of Datamentioning

confidence: 99%

Characterization of radioligand binding to a transmembrane receptor reconstituted into Lipobeads

Park

Buck

et al. 2004

FEBS Letters

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Lipobeads are hydrogel beads surrounded by a lipid bilayer membrane and have been developed to act as a cell analogue. The FLAG-tagged M 2 muscarinic receptor was incorporated onto the surface of the Lipobead by incubating pre-Lipobeads with proteoliposomes containing the receptor. Receptors reconstituted onto the surface of the Lipobeads were functional in that they bound the antagonists quinuclidinylbenzilate and scopolamine with characteristic muscarinic affinities. This demonstrates the feasibility of using Lipobeads to study the binding properties of the M 2 muscarinic receptor and offers a promising approach to the study of transmembrane protein biology in general.

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Cooperativity Manifest in the Binding Properties of Purified Cardiac Muscarinic Receptors

Cited by 159 publications

References 51 publications

Subunits of a Yeast Oligomeric G Protein-coupled Receptor Are Activated Independently by Agonist but Function in Concert to Activate G Protein Heterotrimers

Subunits of a Yeast Oligomeric G Protein-coupled Receptor Are Activated Independently by Agonist but Function in Concert to Activate G Protein Heterotrimers

Oligomeric potential of the M₂ muscarinic cholinergic receptor

Characterization of radioligand binding to a transmembrane receptor reconstituted into Lipobeads

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Cooperativity Manifest in the Binding Properties of Purified Cardiac Muscarinic Receptors

Cited by 159 publications

References 51 publications

Subunits of a Yeast Oligomeric G Protein-coupled Receptor Are Activated Independently by Agonist but Function in Concert to Activate G Protein Heterotrimers

Subunits of a Yeast Oligomeric G Protein-coupled Receptor Are Activated Independently by Agonist but Function in Concert to Activate G Protein Heterotrimers

Oligomeric potential of the M2 muscarinic cholinergic receptor

Characterization of radioligand binding to a transmembrane receptor reconstituted into Lipobeads

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Oligomeric potential of the M₂ muscarinic cholinergic receptor