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Assembly and intracellular targeting of the betagamma subunits of heterotrimeric G proteins.

Rehm A, Ploegh HL - J. Cell Biol. (1997)

Bottom Line: The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent alpha, beta, and gamma subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact.Monoclonal anti-beta antibodies (ARC5 and ARC9), raised against immunoaffinity purified beta gamma complexes, recognize beta subunits when not associated with gamma and can thus be used to monitor assembly of beta gamma complexes.Brefeldin A treatment does not interfere with delivery of beta gamma subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent alpha, beta, and gamma subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-beta antibodies (ARC5 and ARC9), raised against immunoaffinity purified beta gamma complexes, recognize beta subunits when not associated with gamma and can thus be used to monitor assembly of beta gamma complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of beta gamma complexes with the plasma membrane fraction starts between 15-30 min of chase. Three pools of beta subunits can be distinguished based on their association with gamma subunits, their localization, and their detergent solubility. Association of beta and alpha subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of beta gamma subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane.

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βγ complexes are enriched in detergent-resistant membrane domains. (A) IMR-32 cells (5 × 106) were lysed directly in  PBS/1% SDS/2 mM DTT; alternatively an equal amount of cells  was first extracted with TX-100–containing lysis buffer, followed  by re-extraction of the detergent-resistant membrane fraction in  PBS/1% SDS/2 mM DTT. Total cell lysate and TX-100–resistant  pellet were heated twice for 3 min at 100°C in the presence of  SDS, and passed through a 21-gauge needle several times. Laemmli  sample buffer was added and samples were analyzed after SDSPAGE and immunoblot for the presence of G protein β subunits.  (B) Equal numbers of cells were cultured for 16–18 h in the presence (+) or absence (−) of 20% FCS. Total cell lysates and detergent-resistant membrane pellets were generated as described  in A. Samples were normalized for the amount of protein (100  μg/lane) using the BCA protein assay. Samples were analyzed after SDS-PAGE and immunoblot, using the anti-β mAb ARC5.
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Figure 6: βγ complexes are enriched in detergent-resistant membrane domains. (A) IMR-32 cells (5 × 106) were lysed directly in PBS/1% SDS/2 mM DTT; alternatively an equal amount of cells was first extracted with TX-100–containing lysis buffer, followed by re-extraction of the detergent-resistant membrane fraction in PBS/1% SDS/2 mM DTT. Total cell lysate and TX-100–resistant pellet were heated twice for 3 min at 100°C in the presence of SDS, and passed through a 21-gauge needle several times. Laemmli sample buffer was added and samples were analyzed after SDSPAGE and immunoblot for the presence of G protein β subunits. (B) Equal numbers of cells were cultured for 16–18 h in the presence (+) or absence (−) of 20% FCS. Total cell lysates and detergent-resistant membrane pellets were generated as described in A. Samples were normalized for the amount of protein (100 μg/lane) using the BCA protein assay. Samples were analyzed after SDS-PAGE and immunoblot, using the anti-β mAb ARC5.

Mentions: The presence of βγ complexes and other proteins involved in signal transduction in detergent-resistant domains has been attributed physiological significance. It has been argued that these detergent-resistant membranes fulfill a specialized role in signaling by allowing a high local concentration of the relevant proteins. We sought to explore this issue by comparing the relative amounts of βγ in detergent-resistant membranes from cells held under serum starvation vs cells maintained in 20% FCS. For cells cultured in the presence of FCS, we observed that ∼30% of all β subunits could be retrieved in the detergent-resistant membranes, where it must be kept in mind that even non-ionic detergent may extract some βγ's (and other membrane proteins) from areas generally defined as detergent-resistant membranes (Fig. 6 A). When βγ content was analyzed for cells maintained in the presence or absence of serum (normalized for the amount of protein loaded for each sample, Fig. 6 B), we observed an approximately threefold enrichment of βγ's in detergent-resistant membranes from serum-stimulated cells.


Assembly and intracellular targeting of the betagamma subunits of heterotrimeric G proteins.

Rehm A, Ploegh HL - J. Cell Biol. (1997)

βγ complexes are enriched in detergent-resistant membrane domains. (A) IMR-32 cells (5 × 106) were lysed directly in  PBS/1% SDS/2 mM DTT; alternatively an equal amount of cells  was first extracted with TX-100–containing lysis buffer, followed  by re-extraction of the detergent-resistant membrane fraction in  PBS/1% SDS/2 mM DTT. Total cell lysate and TX-100–resistant  pellet were heated twice for 3 min at 100°C in the presence of  SDS, and passed through a 21-gauge needle several times. Laemmli  sample buffer was added and samples were analyzed after SDSPAGE and immunoblot for the presence of G protein β subunits.  (B) Equal numbers of cells were cultured for 16–18 h in the presence (+) or absence (−) of 20% FCS. Total cell lysates and detergent-resistant membrane pellets were generated as described  in A. Samples were normalized for the amount of protein (100  μg/lane) using the BCA protein assay. Samples were analyzed after SDS-PAGE and immunoblot, using the anti-β mAb ARC5.
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Figure 6: βγ complexes are enriched in detergent-resistant membrane domains. (A) IMR-32 cells (5 × 106) were lysed directly in PBS/1% SDS/2 mM DTT; alternatively an equal amount of cells was first extracted with TX-100–containing lysis buffer, followed by re-extraction of the detergent-resistant membrane fraction in PBS/1% SDS/2 mM DTT. Total cell lysate and TX-100–resistant pellet were heated twice for 3 min at 100°C in the presence of SDS, and passed through a 21-gauge needle several times. Laemmli sample buffer was added and samples were analyzed after SDSPAGE and immunoblot for the presence of G protein β subunits. (B) Equal numbers of cells were cultured for 16–18 h in the presence (+) or absence (−) of 20% FCS. Total cell lysates and detergent-resistant membrane pellets were generated as described in A. Samples were normalized for the amount of protein (100 μg/lane) using the BCA protein assay. Samples were analyzed after SDS-PAGE and immunoblot, using the anti-β mAb ARC5.
Mentions: The presence of βγ complexes and other proteins involved in signal transduction in detergent-resistant domains has been attributed physiological significance. It has been argued that these detergent-resistant membranes fulfill a specialized role in signaling by allowing a high local concentration of the relevant proteins. We sought to explore this issue by comparing the relative amounts of βγ in detergent-resistant membranes from cells held under serum starvation vs cells maintained in 20% FCS. For cells cultured in the presence of FCS, we observed that ∼30% of all β subunits could be retrieved in the detergent-resistant membranes, where it must be kept in mind that even non-ionic detergent may extract some βγ's (and other membrane proteins) from areas generally defined as detergent-resistant membranes (Fig. 6 A). When βγ content was analyzed for cells maintained in the presence or absence of serum (normalized for the amount of protein loaded for each sample, Fig. 6 B), we observed an approximately threefold enrichment of βγ's in detergent-resistant membranes from serum-stimulated cells.

Bottom Line: The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent alpha, beta, and gamma subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact.Monoclonal anti-beta antibodies (ARC5 and ARC9), raised against immunoaffinity purified beta gamma complexes, recognize beta subunits when not associated with gamma and can thus be used to monitor assembly of beta gamma complexes.Brefeldin A treatment does not interfere with delivery of beta gamma subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent alpha, beta, and gamma subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-beta antibodies (ARC5 and ARC9), raised against immunoaffinity purified beta gamma complexes, recognize beta subunits when not associated with gamma and can thus be used to monitor assembly of beta gamma complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of beta gamma complexes with the plasma membrane fraction starts between 15-30 min of chase. Three pools of beta subunits can be distinguished based on their association with gamma subunits, their localization, and their detergent solubility. Association of beta and alpha subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of beta gamma subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane.

Show MeSH
Related in: MedlinePlus