Role of G Protein βγ Complex in Receptor–G Protein Interaction

Azpiazu, Inaki; Gautam, N.

doi:10.1016/s0076-6879(02)44709-x

Cited by 13 publications

(13 citation statements)

References 21 publications

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“…Purification of M2, reconstitution of the receptor and receptor stimulated GTPase assays were performed as described previously [22]. Briefly, His-tagged M2 receptor was expressed in insect cells, purified and reconstituted into brain lipids.…”

Section: Synthesis and Purification Of M2 Receptor G Protein Subunitmentioning

confidence: 99%

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

Akgöz¹,

Kalyanaraman²,

Gautam³

2006

Cellular Signalling

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On activation of a receptor the G protein βγ complex translocates away from the receptor on the plasma membrane to the Golgi complex. The rate of translocation is influenced by the type of γ subunit associated with the G protein. Complementary approaches -imaging living cells expressing fluorescent protein tagged G proteins and assaying reconstituted receptors and G proteins in vitrowere used to identify mechanisms at the basis of the translocation process. Translocation of Gβγ containing mutant γ subunits with altered prenyl moieties showed that the differences in the prenyl moieties were not sufficient to explain the differential effects of geranylgeranylated γ5 and farnesylated γ11 on the translocation process. The translocation properties of Gβγ were altered dramatically by mutating the C terminal tail region of the γ subunit. The translocation characteristics of these mutants suggest that after receptor activation, Gβγ retains contact with a receptor through the γ subunit C terminal domain and that differential interaction of the activated receptor with this domain controls Gβγ translocation from the plasma membrane.

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Section: Synthesis and Purification Of M2 Receptor G Protein Subunitmentioning

confidence: 99%

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

Akgöz¹,

Kalyanaraman²,

Gautam³

2006

Cellular Signalling

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“…RGS4 acts as a GTPase-activating protein for the G o/i family (14). RGS4 potentiated M2-stimulated GTPase activity over 10-fold (7). When the time course of M2-stimulated GTPase activity was measured in the presence of RGS4, the M2 receptor activated ␣o␤1␥5scr significantly more than the wild type (Fig.…”

Section: Effect Of Mutating the C-terminal Domain Of The ␥5 Subunit Omentioning

confidence: 90%

“…Cys residues were retained at the C terminus for chemical geranylgeranylation using geranylgeranyl bromide. Prenylation and purification using fast protein liquid chromatography has been described (7). The integrity of the modified peptides were checked by mass spectrometry and chromatography, and peptide concentrations were estimated by amino acid analysis.…”

Section: Synthesis and Prenylation Of Peptides-mentioning

confidence: 99%

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

Azpiazu

Gautam

2001

Journal of Biological Chemistry

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The surfaces of heterotrimeric G proteins (␣␤␥) in contact with receptors and the molecular events at these sites, which lead to G protein activation, are largely unknown. We show here that a peptide from the C terminus of a G protein ␥ subunit blocks muscarinic receptor-stimulated G protein activation in a sequencedependent fashion. A G protein mutated at the same site on the ␥ subunit shows enhanced receptor stimulated nucleotide exchange without affecting G protein heterotrimerization. Ineffective contact between the ␥ subunit and receptor increases the rate of receptor-stimulated nucleotide exchange. Specific interaction of the G protein ␥ subunit with the receptor thus helps the ␤␥ complex to act at a distance and control guanine nucleotide exchange in the ␣ subunit.Although G protein signaling is central to a vast majority of pathways that control the physiology of mammalian cells, the coordinated changes in the conformations of the agonist bound receptor and the G protein subunits that trigger nucleotide release are not known. Peptide and mutant studies using biochemical assays that measure receptor-G protein coupling have implicated three protein domains, the N-and C-terminal domains of the ␣ subunit and the C-terminal domain of the ␥ subunit in receptor interaction (1-3). Functional contact between the ␥ subunit and a receptor is one mechanism that can explain the universal requirement of the ␤␥ complex for receptor-mediated nucleotide exchange in the ␣ subunit. However, the particular role that the ␥ subunit plays at the receptor surface during G protein activation has not been clear. The crystal structure of the G protein heterotrimer indicates that the C termini of the ␥ and ␣ subunits are a considerable distance from the nucleotide-binding site in the ␣ subunit (4, 5). The mechanisms that help the receptor regulate nucleotide exchange by contacting these domains are therefore an outstanding puzzle.Because a peptide specific to the C terminus of the ␥1 subunit type stabilizes activated rhodopsin in a sequence-dependent manner (6) and a homologous ␥5 peptide specifically inhibits muscarinic receptor modulation of a Ca 2ϩ current in intact neurons (3), we examined the effect of a geranylgeranylated ␥5 peptide on the M2 muscarinic receptor activation of a G protein. The peptide specific to the C-terminal 14 residues of the ␥5 subunit was prenylated because the native ␥5 subunit is modified with geranylgeranyl at the C-terminal Cys residue that is part of a CAAX motif (2). This peptide inhibited activation of G proteins by reconstituted M2 receptor. A peptide with the same sequence scrambled was inactive. These studies indicated that the ␥ subunit peptide interacts with the receptor in a sequence-specific fashion. To test the effect of mutations in this C-terminal domain of ␥5 on G protein activation by M2, C-terminal residues of ␥5 corresponding to the peptides were scrambled. The scrambled sequence was identical to the sequence of the scrambled peptide used in the earlier experiments. Wild type and mut...

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“…Preparation of Purified and Reconstituted Recombinant M2-A detailed description of M2 purification, reconstitution, and measurement of G protein stimulation has been published elsewhere (31). His 6 -tagged M2 was expressed in Sf9 cells by using the recombinant baculovirus (kind gift from Dr. E. Ross).…”

Section: Materials-pipmentioning

confidence: 99%

G Protein β Subunit Types Differentially Interact with a Muscarinic Receptor but Not Adenylyl Cyclase Type II or Phospholipase C-β2/3

Hou¹,

Chang²,

Capper

et al. 2001

Journal of Biological Chemistry

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In comparison with the ␣ subunit of G proteins, the role of the ␤ subunit in signaling is less well understood. During the regulation of effectors by the ␤␥ complex, it is known that the ␤ subunit contacts effectors directly, whereas the role of the ␤ subunit is undefined in receptor-G protein interaction. Among the five G protein ␤ subunits known, the ␤ 4 subunit type is the least studied. We compared the ability of ␤␥ complexes containing ␤ 4 and the well characterized ␤ 1 to stimulate three different effectors: phospholipase C-␤2, phospholipase C-␤3, and adenylyl cyclase type II. ␤ 4 ␥ 2 and ␤ 1 ␥ 2 activated all three of these effectors with equal efficacy. However, nucleotide exchange in a G protein constituting ␣ o ␤ 4 ␥ 2 was stimulated significantly more by the M2 muscarinic receptor compared with ␣ o ␤ 1 ␥ 2 . Because ␣ o forms heterotrimers with ␤ 4 ␥ 2 and ␤ 1 ␥ 2 equally well, these results show that the ␤ subunit type plays a direct role in the receptor activation of a G protein.The G protein ␤␥ complex regulates the activity of a diverse set of effectors, including phospholipases, adenylyl cyclases, and ion channels (1). There is evidence that the ␤ subunit in the complex interacts directly with effectors (2-5). There are five ␤ subtypes (␤ 1 -␤ 5 ) as well as an alternatively spliced version of ␤ 5 (known as ␤ 5 -long) (6 -11). ␤ 1 -␤ 4 share over 80% identity with one another, whereas ␤ 5 shares only ϳ50% identity with the other ␤ subunits (12). The divergence between ␤ 5 and the other ␤ subunits is consistent with the functional differences between ␤ 5 and ␤ 1 observed in effector regulation in a variety of systems (4,13,14). The high sequence similarity of ␤ 1 -␤ 4 suggests that their functions are conserved. Although some experiments indicated little difference in effector modulating capability among these ␤ subunit types, other experiments suggest otherwise. The G protein-coupled receptor kinase GRK3 binds ␤␥ complexes consisting of ␤ 1 , ␤ 2 , and ␤ 3 , but only ␤ 1 and ␤ 2 bind to the related kinase GRK2 (15). Other results indicate the selective mediation of cross-talk between G proteins and protein kinase C modulation of N-type channels by the ␤ 1 subunit type (16).Experiments focusing on the specific role of individual G protein subunit types have provided evidence for a certain level of selectivity in the interaction of ␣ subunit types with receptors (17). Evidence for similar selectivity of interaction between ␥ subunit types and receptors also exists (18 -20). In contrast there is limited evidence for ␤ subunit type selectivity in receptor interaction. Whole-cell experiments using antisense oligonucleotides directed against specific ␤ subunit cDNAs selectively disrupted signaling from particular receptors (21). Although the selective interaction of ␤ subunit types with receptors could give rise to this result, such selectivity has not been shown so far.Among the five ␤ subunits, ␤ 4 is the least studied. Its role in effector regulation and receptor interaction remains unclear. To exam...

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Role of G Protein βγ Complex in Receptor–G Protein Interaction

Cited by 13 publications

References 21 publications

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

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

G Protein β Subunit Types Differentially Interact with a Muscarinic Receptor but Not Adenylyl Cyclase Type II or Phospholipase C-β2/3

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