G Protein γ Subunit Interaction with a Receptor Regulates Receptor-stimulated Nucleotide Exchange

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104

Fluorescence recovery after photobleaching of muscarinic receptors and G protein subunits tagged with cyan or yellow fluorescent protein showed that receptors and G proteins were mobile and not immobilized on the cell membrane. The cyan fluorescent protein-tagged G␣ and yellow fluorescent protein-tagged G␤ subunits were used to develop sensors that coupled selectively with the M2 and M3 muscarinic receptors. In living Chinese hamster ovary cells, imaging showed that sensors emitted a fluorescence resonance energy transfer signal that was abrogated on receptor activation. When sequentially activated with highly expressed muscarinic receptors and endogenous receptors expressed at low levels, sensor molecules were sensitive to the sequence of activation and the receptor numbers. The results distinguish between models proposing that receptor and G protein types interact freely with each other on the cell membrane or that they function as mutually exclusive multimolecular complexes by providing direct support for the former model in these cells.G proteins and their receptors have been studied extensively, but it is still not known whether they are immobilized at certain locations on the cell membrane or are capable of diffusing to various regions on the cell membrane. By tagging a muscarinic receptor, a G␣ subunit, and the G␤␥ complex with CFP 1 or YFP and examining living cells with fluorescence recovery after photobleaching, we have determined that the receptor and G protein subunits are mobile. The mobility of the signaling molecules, however, did not indicate whether particular receptors and G proteins occur in mutually exclusive multimolecular complexes or freely diffuse to collide with each other. A longstanding model proposes that specificity is achieved by mechanisms that allow certain receptor types and G protein types to be associated in exclusive multimolecular complexes (1, 2). It has been proposed that receptors and G proteins exist as complexes even in the absence of an agonist stimulus and that G protein activation occurs in isolation from other such molecular assemblies (2, 3). In a further extension of this model, it has been suggested that receptor-G protein complexes persist during signaling activity (4). These models would explain how mammalian cells respond to extracellular signals with specificity, although they often express a variety of different receptor and G protein subunit types (5). Such a mechanism would aid the rapid response to the initial signal because of the association of appropriate receptor and G protein types before activation. Evidence for the presence of assemblies of receptors and G proteins in cell membranes and their potential interaction with adaptor or scaffolding proteins have also been thought to promote such receptor-G protein molecular assemblies (4, 6 -9).An alternative model predicts that receptor types and G protein types are mobile on the cell membrane, colliding with each other freely (1, 10). For optimal determination of which of these mechanisms regulate recept...

Section: Resultsmentioning

confidence: 99%

A Fluorescence Resonance Energy Transfer-based Sensor Indicates that Receptor Access to a G Protein Is Unrestricted in a Living Mammalian Cell

Azpiazu

Gautam

2004

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104

“…Later studies have identified several regions on the ␤ subunit that interact with effectors divided into regions that are involved in stabilizing interaction and others involved in modulating effector activity (3)(4)(5)(6)(7)(8). Although there is increasing evidence that the G protein ␥ subunits specify contact with receptors (9,10), their role in effector regulation has been less clear. Early evidence indicated that the prenyl modification at the C terminus of the ␥ subunit is a requirement for the ␤␥ complex action on effectors (11).…”

mentioning

confidence: 99%

“…A third mutant has 10 residues upstream of the Cys residue deleted, thus shortening the Cterminal tail domain. Ten residues were removed because this domain has previously been shown to be the minimum sequence critical for receptor interaction of the G protein heterotrimer (9,10,14). A fourth mutant contains an Ala substituted for Phe at position 59.…”

mentioning

confidence: 99%

Role of the G Protein γ Subunit in βγ Complex Modulation of Phospholipase Cβ Function

Akgöz¹,

Azpiazu²,

Kalyanaraman³

et al. 2002

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The G protein ␤␥ complex regulates a wide range of effectors, including the phospholipase C isozymes (PLC␤s). Different domains on the ␤ subunit are known to contact phospholipase C␤ and affect its regulation. In contrast, the role of the ␥ subunit in G␤␥ modulation of PLC␤ function is not known. Results here show that the ␥ subunit C-terminal domain is involved in mediating G␤␥ interactions with phospholipase C␤. Mutations were introduced to alter the position of the post-translational prenyl modification at the C terminus of the ␥ subunit with reference to the ␤ subunit. These mutants were appropriately post-translationally modified with the geranylgeranyl moiety. A deletion that shortened the C-terminal domain, insertions that extended this domain, and a point mutation, F59A, that disrupted the interaction of this domain with the ␤ subunit were all affected in their ability to activate PLC␤ to varying degrees. All mutants, however, interacted equally effectively with the G o ␣ subunit. The results indicate that the G protein ␥ subunit plays a direct role in the modulation of effector function by the ␤␥ complex.The G protein ␤␥ complex modulates the function of a number of effectors (1). The precise molecular mechanisms underlying the activation or inhibition of an effector by the G protein ␤␥ complex is not clear. Regions on the ␤ subunit and effectors important for interaction between G␤␥ and effector molecules have been identified. A domain on the G protein ␤ subunit was initially shown to contact domains on several effector molecules (2). Later studies have identified several regions on the ␤ subunit that interact with effectors divided into regions that are involved in stabilizing interaction and others involved in modulating effector activity (3-8). Although there is increasing evidence that the G protein ␥ subunits specify contact with receptors (9, 10), their role in effector regulation has been less clear. Early evidence indicated that the prenyl modification at the C terminus of the ␥ subunit is a requirement for the ␤␥ complex action on effectors (11). More recently, it has been shown that if the type of prenylation on ␥ subunits, farnesyl (C15) or geranylgeranyl (C20), is altered through mutational alteration of the protein, the ability of the mutant ␤␥ complex to act on effectors is altered (12). These results confirmed the role for the prenyl moiety in effector activation. The reasons for the requirement of the prenyl modification were, however, not clear. It was unclear whether the prenyl group was required because it assisted the ␤␥ complex association with membranes, and as a consequence, brought the ␤␥ complex in close proximity to membrane-bound effector molecules or whether the prenyl group actually interacted with effectors and stabilized this critical protein-protein interaction event. Recent evidence supports the latter mechanism, indicating that the prenyl moiety physically contacts at least one effector, PLC␤, 1 and facilitates ␤␥ interaction with PLC␤ (13).To further define the role o...

“…Although the guanine nucleotide binding site and GTPase machinery are defined by the G protein ␣ subunit, the ␤/␥ complex plays a key role, with growing evidence for a direct role of the ␥ subunit in contacting the receptor to enhance guanine nucleotide exchange (15,16). By mutating a key ␤/␥ contact site in G␣ 11 we also provide support for this notion, because the modified ␣ subunit exchanges guanine nucleotide significantly less effectively in response to receptor activation that does the wild type.…”

mentioning

confidence: 99%

Effective Information Transfer from the α1b-Adrenoceptor to Gα11 Requires Both β/γ Interactions and an Aromatic Group Four Amino Acids from the C Terminus of the G Protein

Liu

Carrillo

Pediani

et al. 2002

Co-expression of the ␣ 1b -adrenoreceptor and G␣ 11 in cells derived from a G␣ q /G␣ 11 knock-out mouse allows agonistmediated elevation of intracellular Ca 2؉ levels that is transduced by ␤/␥ released from the G protein ␣ subunit. Mutation of Tyr 356 of G␣ 11 to Phe, within a receptor contact domain, had little effect on function but this was reduced greatly by alteration to Ser and virtually eliminated by conversion to Asp. This pattern was replicated following incorporation of each form of G␣ 11 into fusion proteins with the ␣ 1b -adrenoreceptor. Following a [ 2؉ levels, as did a mutation in G␣ 11 that prevents G protein subunit dissociation. These results indicate that a bulky aromatic group is required four amino acids from the C terminus of G␣ 11 to maximize information transfer from an agonist-occupied receptor and disprove the hypothesis that tyrosine phosphorylation of this residue is required for G protein activation (Umemori, H