Structural heterogeneity of membrane receptors and GTP‐binding proteins and its functional consequences for signal transduction

Boege, Fritz; Neumann, Eberhard; Helmreich, Ernst

doi:10.1111/j.1432-1033.1991.tb16085.x

Cited by 42 publications

(11 citation statements)

References 253 publications

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“…Heterotrimeric G proteins consist of cc, j3, and y subunits [4] and are activated by seven transmembrane spanning receptors [5,6]. Binding of ligand to receptor causes exchange of GDP for GTP on the a subunit which induces a conformational change in the heterocomplex and subsequent activation of the subunits [2,5].…”

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confidence: 99%

Rapid kinetics of G protein subunit association: A rate‐limiting conformational change?

Neubig¹,

Connolly²,

Remmers³

1994

FEBS Letters

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G protein subunit association and dissociation are thought to play an important role in signal transduction. We measured a@7 heterocomplex formation using resonance energy transfer. Fluorescein-labelled @F-a) emission was quenched _ 10% on mixing with eosin-labelled /37(E$7).Unlabelled b7 did not quench F-a fluorescence. Stopped-flow kinetics showed a t In ranging from 2.5 s to 0.25 s for 50 nM to 1200 nM E-jI7. The rate saturated at high E-p7 concentrations consistent with a two-step mechanism. We report the first rapid-mix studies of G protein subunit association kinetics which suggest that a and /I7 combine by a two-step process with a maximal rate of 4.1 + 0.4 s-'.Key words: Resonance energy transfer; Rapid kinetics; Fluorescence rntroductionG proteins transmit signals to many effcctors including adenylyl cyclase, K+ and Ca" channels, and phosphodiesterases [l-3]. Heterotrimeric G proteins consist of cc, j3, and y subunits [4] and are activated by seven transmembrane spanning receptors [5,6]. Binding of ligand to receptor causes exchange of GDP for GTP on the a subunit which induces a conformational change in the heterocomplex and subsequent activation of the subunits [2,5]. The heterocomplex is thought to dissociate into a and & subunits each capable of mediating cellular responses [2,7-91. While dissociation is well documented in detergent solutions it has been hard to examine in natural or synthetic membranes.To better understand the physical interactions of G protein subunits during activation and deactivation we used fluorescent G protein subunits. With different fluorophores on the a and By subunits, subunit interactions can be followed in real time by resonance energy transfer (RET). We report the first rapid kinetic analysis of G protein subunit association and propose a two-step model for association. An initial pre-equilibrium interaction occurs with an affinity -300 nM, followed by the appearance of a high affinity interaction occurring with a maximum rate of -4 s-r. This conformational change may limit the rate of deactivation of G protein a subunits after GTP hydrolysis in the physiological setting. Materials and methods MaterialsFluorescein 5-isothiocyanate (FITC) and eosin-5-isothiocyanate (EEITC) were uurchased from Molecular Probes. Inc. (Euaene. OR). $5S]GTPyS was obtained from New England Nuclear (Boston; MA). Other reagents were from standard suppliers. Fhwescent subunit preparationFluorescein-and eosin-labelled G, were prepared essentially as de- . Singly labelled F-G, was isolated by Mono-Q chromatography. Free eosin was removed from eosin-labelled-G, by gel tiltration on Sephadex G-50 and Centricon 30 ultrafiltration. Individual labelled subunits F-u and E-/I7 were resolved by activation in AMF (10 mM MgC&, 10 mM NaF, 20 PM AlClJ in TEDN buffer (10 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, 100 mM NaCl) containing 0.3% sodium cholate followed by heptylamine Sepharose chromatography in a sodium chloride and cholate gradient and storage in aliquots at -70°C for several weeks prior to us...

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confidence: 99%

Rapid kinetics of G protein subunit association: A rate‐limiting conformational change?

Neubig¹,

Connolly²,

Remmers³

1994

FEBS Letters

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“…This interaction results in two important events: (1) the replacement of GDP on the a subunit by GTP and (2) the dissociation of the heterotrimer into a Py complex plus a free a subunit. The latter is then capable of Ross, 1989;Birnbaumer, 1990;Boege et al, 1991). Two types of related results have primarily led to this conclusion.…”

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confidence: 90%

“…Other related receptors in this family include receptors for glucagon, TSH, and angiotensin II (Bimbaumer, 1990). All G proteincoupled receptors have common structural features (e.g., Kerlavage et al, 1987;Bonner, 1989;Lechleiter et al, 1990;Boege et al, 1991). The similarities include the following: seven transmembrane a-helical domains, an extracellular N-terminal region, and an intracellular C-terminal region.…”

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confidence: 99%

“…The first is the demonstration of cross talk (interactions) between two signaling systems, that is, one receptor pathway influencing another. For example, we have suggested that in the rat parotid acinar cell the 3-adrenergic receptor via cAMP can elicit inositol trisphosphate (IP3) formation and subsequent Ca2+ mobilization (Hom et al, 1989 (Ross, 1989;Bimbaumer, 1990;Boege, et al, 1991; Figure 1). An obvious corollary of these findings is that it is inappropriate to generalize broadly about signal routing from one cell type to another, even if identical neurotransmitter stimuli are involved.…”

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confidence: 99%

“…This article, will focus primarily on the G protein and discuss signaling (particularly that which is relevant to saliva formation) from the viewpoint of receptor and effector protein interactions with the G protein. There are many relatively recent general reviews on G proteins and their function to which the reader is referred for more details (e.g., Freissmuth et al, 1989;Ross, 1989;Bimbaumer, 1990;Boege et al 1991).…”

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confidence: 99%

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Signaling Mechanisms That Regulate Saliva Formation

Baum

Dai

Hiramatsu

et al. 1993

Critical Reviews in Oral Biology & Medicine

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The precipitating event in the formation of saliva is the binding of neurotransmitter molecules to cell surface receptor proteins. The principal neurotransmitters involved are acetylcholine and norepinephrine that bind, respectively, to muscarinic-cholinergic, and a-and ,-adrenergic receptors. The transduction of the extracellular signal requires an integral membrane protein capable of binding GTP, a G protein, that specifically interacts with the receptor. The components of G protein transduction systems are fairly well studied, but the pathways by which signals are routed are just being recognized. Delineation of such routing pathways is essential to understanding the regulation of saliva formation.

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Bioamine receptors: Evolutionary and functional variations of a structural leitmotiv

Vernier¹,

Piégay²,

Samama³

et al. 1993

Comparative Molecular Neurobiology

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Structural heterogeneity of membrane receptors and GTP‐binding proteins and its functional consequences for signal transduction

Cited by 42 publications

References 253 publications

Rapid kinetics of G protein subunit association: A rate‐limiting conformational change?

Rapid kinetics of G protein subunit association: A rate‐limiting conformational change?

Signaling Mechanisms That Regulate Saliva Formation

Bioamine receptors: Evolutionary and functional variations of a structural leitmotiv

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