Effect of monoclonal antibody binding on .alpha.-.beta..gamma. subunit interactions in the rod outer segment G protein, Gt

Mazzoni, Maria Rosa; Hamm, Heidi E.

doi:10.1021/bi00451a047

Cited by 26 publications

(18 citation statements)

References 36 publications

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“…Although a second surface of the ␣ subunit forms an even larger area of interaction with ␤␥ (33, 34), our studies concentrated on the N-terminal region for two important reasons. First, previous work by others showed that disruption of this region by deletion (36,37), proteolysis (38), or antibody binding (39,40) is sufficient to prevent ␣ from interacting with ␤␥. Second, mutations in the N-terminal region are unlikely to disrupt the overall three-dimensional structure of the ␣ subunit.…”

Section: Mutation Of a Conserved ␤␥ Contact Region In ␣ S And ␣ Q -mentioning

confidence: 99%

Interaction with Gβγ Is Required for Membrane Targeting and Palmitoylation of Gαs and Gαq

Evanko

Thiyagarajan

Wedegaertner

2000

Journal of Biological Chemistry

115

158

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Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein alpha subunits. For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein betagamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to betagamma were mutated to create a series of betagamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy. The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional alpha(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation. These findings identify binding to betagamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of betagamma binding-deficient alpha(q) subunits.

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Section: Mutation Of a Conserved ␤␥ Contact Region In ␣ S And ␣ Q -mentioning

confidence: 99%

Interaction with Gβγ Is Required for Membrane Targeting and Palmitoylation of Gαs and Gαq

Evanko

Thiyagarajan

Wedegaertner

2000

Journal of Biological Chemistry

115

158

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“…Numerous biochemical (29,31,47,48) and mutational (49,50) studies have implicated the amino terminus of G protein ␣ subunits in the interaction with ␤␥ subunits. The resolution of the crystal structure of heterotrimeric G t (14) has confirmed that the amino-terminal helix of the ␣ t subunit is involved in forming a binding site for the ␤ t subunit.…”

Section: Fig 3 Identification Of ␣ T Proteolytic Fragments By Immunmentioning

confidence: 99%

Interaction of Transducin with Light-activated Rhodopsin Protects It from Proteolytic Digestion by Trypsin

Mazzoni

Hamm

1996

Journal of Biological Chemistry

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The tryptic cleavage pattern of transducin (G t ) in solution was compared with that in the presence of phospholipid vesicles, rod outer segment (ROS) membranes kept in the dark, or ROS membranes containing lightactivated rhodopsin, metarhodopsin II (Rh*). When G t was in the high affinity complex with Rh*, the ␣ t subunit was almost completely protected from proteolysis. , only a few angstroms away from the carboxyl terminus of ␣ t , which is known to directly bind to Rh*, is likely to also be a part of the Rh* binding site. This is in agreement with other studies and has implications for the mechanism by which receptors catalyze GDP release from G proteins. The protection of Lys 18 in the presence of phospholipid vesicles suggests that the amino-terminal region is in contact with the membrane, consistent with the crystal structure of the heterotrimer (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319).Certain extracellular signals, including hormones, neurotransmitters, neuromodulators, chemokines, odorants, and light, activate a class of receptors that initiate cellular effects via activation of heterotrimeric G proteins. Agonist binding to the G protein-coupled receptors leads to conformational changes that promote a tighter interaction with specific G proteins, catalysis of GDP release, and subsequent G protein activation. In the absence of guanine nucleotides, agonist binding to the receptor is stabilized by the bound G protein. The structural basis of the ternary complex among agonist, receptor, and heterotrimeric G protein is an active area of study. Extensive mutagenesis experiments, as well as peptide competition investigations for a variety of G protein-coupled receptors, have led to an understanding that the second and third cytoplasmic loops and, in some circumstances, the putative fourth loop, as well as portions of ␣ helices VI and VII, are important in recognition of cognate G proteins (1-3). It has been shown that the heterotrimeric G protein, rather than just the ␣ or ␤␥ subunits, is required for the interaction, but studies pointing out the importance of specific regions are thus far limited to the ␣ subunits (4 -7).The crystal structures of the active (GTP␥S 1 and GDP AlF 4 Ϫ -bound) (8 -10) and inactive (GDP-bound) (11, 12) forms of the ␣ subunits of transducin (G t ) and G i1 have been reported. Analysis of the two crystal forms has established the nature of the conformational change induced by the exchange of GTP for GDP and the switch mechanism by which the presence or absence of the ␥-phosphate defines the active or inactive state of the ␣ t subunit (9, 11). The high resolution crystal structures of the heterotrimeric G proteins, G t and G i1 , provide a fundamental context for understanding how a heterotrimer interacts with the membrane and with activated receptors (13,14). The molecular mechanisms involved in the conformational changes of the ␣ subunit and the nucleotide exchange induced by the heterotrimeric G protein inter...

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“…As B binds independently to P 1 and P 2 , the binding constants (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) can be obtained: …”

Section: :1 and 2:1 Competitive Binding Stoichiometrymentioning

confidence: 99%

“…(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) shows that Eq. (3-17) is consistent with the definition of binding constant of B and P 1 , as shown in the Eq.…”

Section: :1 and 2:1 Competitive Binding Stoichiometrymentioning

confidence: 99%

“…Qualitative and quantitative study of such binding is essential not only to the understanding of the molecular basis for immune functions, and also important to the method development of immunoassays. Various techniques have been developed for binding study, such as enzyme-linked immunosorbent assay (ELISA) [2][3][4], surface plasmon resonance (SPR) [5][6][7], gel electrophoresis mobility shift assay (EMSA) [8,9], and affinity chromatography [10,11]. Most immunoassays heavily rely on the adsorption of antigen or antibody on solid/liquid surface, e.g.…”

Section: Introductionmentioning

confidence: 99%

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Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N2-dG adducts by capillary electrophoresis immunoassay

Wang

et al. 2010

Journal of Chromatography A

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a b s t r a c tThe stereospecific binding of monoclonal antibody (mAb) 8E11 to anti-benzo(a)pyrene diol epoxide (BPDE)-dG adducts in single nucleoside, long oligonucleotide, and genomic DNA were quantitatively evaluated using noncompetitive and competitive capillary electrophoresis (CE) immunoassays. Two single-stranded TMR-BPDE-90mers containing a single anti-BPDE-dG adduct with defined stereochemistry and a fluorescent label at 5 -end were used as fluorescent probes for competitive CE immunoassay. To quantitatively evaluate the binding affinity through competitive CE immunoassays, a series of equations were derived according to the binding stoichiometry. The binding of mAb 8E11 to trans-(+)-anti-BPDE-dG displays strongest affinity (K b : 3.57 × 10 8 M −1 ) among all four investigated anti-BPDE-dG mononucleoside adducts, and the cis-(−)-anti-BPDE-dG displays lowest affinity (K b : 1.14 ×10 7 M −1 ). The binding of monoclonal antibody (mAb) 8E11 to BPDE-dG adducts in long DNA (90mer) preferentially forms the complex with a stoichiometry of 1:1, and that mAb 8E11 displays a slightly higher affinity with trans-(+)-anti-BPDE90mers (K b : 6.36 ± 0.54 × 10 8 M −1 ) than trans-(−)-anti-BPDE-90mers (K b : 4.52 ± 0.52 × 10 8 M −1 ). The mAb 8E11 also displays high affinity with BPDE-dG adducts in genomic DNA (K b : 3.74 × 10 8 M −1 ), indicating its promising applications for sensitive immuno-detection of BPDE-DNA adducts in genomic DNA.

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Effect of monoclonal antibody binding on .alpha.-.beta..gamma. subunit interactions in the rod outer segment G protein, Gt

Cited by 26 publications

References 36 publications

Interaction with Gβγ Is Required for Membrane Targeting and Palmitoylation of Gαs and Gαq

Interaction with Gβγ Is Required for Membrane Targeting and Palmitoylation of Gαs and Gαq

Interaction of Transducin with Light-activated Rhodopsin Protects It from Proteolytic Digestion by Trypsin

Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N2-dG adducts by capillary electrophoresis immunoassay

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