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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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The generation of the βγ subunit derived 27-kD tryptic  cleavage product in cell lysates is coincident with the appearance  of newly assembled βγ complexes. (A) IMR-32 cells were labeled  with 200 μCi [35S]methionine for 2 h. Cells were lysed in 0.3%  Lubrol containing lysis buffer and trypsin was added at the concentrations indicated. Immunoprecipitations were done in the  presence of 0.2% SDS using ARC9 or rabbit anti-βγ serum. The  positions of undigested β chains and tryptic fragments are indicated on the right. (B) The origin of the 27-kD tryptic β fragment  was confirmed by re-immunoprecipitation of ARC9 immunoprecipitates with the peptide-specific polyclonal antiserum U-49. (C)  IMR-32 cells were pulsed for 2 min with 500 μCi [35S]methionine  and chased for the times indicated. Lysates were prepared as in  A, and trypsin was added at a concentration of 50 μg/ml. Immunoprecipitations were done in the presence and absence of 0.2%  SDS. Note that the appearance of the 27-kD fragment is coincident with the loss of the (−) SDS form in untreated samples. (D)  β and γ subunits are complexed in non-ionic detergent-resistant  membrane domains. IMR-32 cells were labeled for 3 h with 150  μCi [35S]methionine and lysed in TX-100–containing lysis buffer.  Detergent-resistant membranes were resolubilized in octyl-glucoside containing lysis buffer. Trypsin digestion was done as described  before (A–C); immunoprecipitations with ARC9 and rabbit antiβγ serum resulted in the appearance of the 27-kD and 14-kD  tryptic fragments.
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Figure 2: The generation of the βγ subunit derived 27-kD tryptic cleavage product in cell lysates is coincident with the appearance of newly assembled βγ complexes. (A) IMR-32 cells were labeled with 200 μCi [35S]methionine for 2 h. Cells were lysed in 0.3% Lubrol containing lysis buffer and trypsin was added at the concentrations indicated. Immunoprecipitations were done in the presence of 0.2% SDS using ARC9 or rabbit anti-βγ serum. The positions of undigested β chains and tryptic fragments are indicated on the right. (B) The origin of the 27-kD tryptic β fragment was confirmed by re-immunoprecipitation of ARC9 immunoprecipitates with the peptide-specific polyclonal antiserum U-49. (C) IMR-32 cells were pulsed for 2 min with 500 μCi [35S]methionine and chased for the times indicated. Lysates were prepared as in A, and trypsin was added at a concentration of 50 μg/ml. Immunoprecipitations were done in the presence and absence of 0.2% SDS. Note that the appearance of the 27-kD fragment is coincident with the loss of the (−) SDS form in untreated samples. (D) β and γ subunits are complexed in non-ionic detergent-resistant membrane domains. IMR-32 cells were labeled for 3 h with 150 μCi [35S]methionine and lysed in TX-100–containing lysis buffer. Detergent-resistant membranes were resolubilized in octyl-glucoside containing lysis buffer. Trypsin digestion was done as described before (A–C); immunoprecipitations with ARC9 and rabbit antiβγ serum resulted in the appearance of the 27-kD and 14-kD tryptic fragments.

Mentions: We establish the occurrence of both characteristic fragments after tryptic digest of IMR-32 cell lysates using mAb ARC9 and the polyclonal rabbit anti-βγ serum (Fig. 2 A). Although we tested different concentration ranges of trypsin, we observed in these experiments the persistence of full-length forms of β, indicating that different subtype combinations of β and γ in IMR-32 cells may exist with different degrees of trypsin susceptibility, or that at least some fraction of βγ may not be fully accessible to trypsin, unlike purified βγ subunits. Re-immunoprecipitation of the 27-kD fragment with the peptide specific polyclonal antibody U-49 (derived against amino acids 131-145 of β1) established the origin of this fragment as β subunit-derived (Fig. 2 B).


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

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

The generation of the βγ subunit derived 27-kD tryptic  cleavage product in cell lysates is coincident with the appearance  of newly assembled βγ complexes. (A) IMR-32 cells were labeled  with 200 μCi [35S]methionine for 2 h. Cells were lysed in 0.3%  Lubrol containing lysis buffer and trypsin was added at the concentrations indicated. Immunoprecipitations were done in the  presence of 0.2% SDS using ARC9 or rabbit anti-βγ serum. The  positions of undigested β chains and tryptic fragments are indicated on the right. (B) The origin of the 27-kD tryptic β fragment  was confirmed by re-immunoprecipitation of ARC9 immunoprecipitates with the peptide-specific polyclonal antiserum U-49. (C)  IMR-32 cells were pulsed for 2 min with 500 μCi [35S]methionine  and chased for the times indicated. Lysates were prepared as in  A, and trypsin was added at a concentration of 50 μg/ml. Immunoprecipitations were done in the presence and absence of 0.2%  SDS. Note that the appearance of the 27-kD fragment is coincident with the loss of the (−) SDS form in untreated samples. (D)  β and γ subunits are complexed in non-ionic detergent-resistant  membrane domains. IMR-32 cells were labeled for 3 h with 150  μCi [35S]methionine and lysed in TX-100–containing lysis buffer.  Detergent-resistant membranes were resolubilized in octyl-glucoside containing lysis buffer. Trypsin digestion was done as described  before (A–C); immunoprecipitations with ARC9 and rabbit antiβγ serum resulted in the appearance of the 27-kD and 14-kD  tryptic fragments.
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Figure 2: The generation of the βγ subunit derived 27-kD tryptic cleavage product in cell lysates is coincident with the appearance of newly assembled βγ complexes. (A) IMR-32 cells were labeled with 200 μCi [35S]methionine for 2 h. Cells were lysed in 0.3% Lubrol containing lysis buffer and trypsin was added at the concentrations indicated. Immunoprecipitations were done in the presence of 0.2% SDS using ARC9 or rabbit anti-βγ serum. The positions of undigested β chains and tryptic fragments are indicated on the right. (B) The origin of the 27-kD tryptic β fragment was confirmed by re-immunoprecipitation of ARC9 immunoprecipitates with the peptide-specific polyclonal antiserum U-49. (C) IMR-32 cells were pulsed for 2 min with 500 μCi [35S]methionine and chased for the times indicated. Lysates were prepared as in A, and trypsin was added at a concentration of 50 μg/ml. Immunoprecipitations were done in the presence and absence of 0.2% SDS. Note that the appearance of the 27-kD fragment is coincident with the loss of the (−) SDS form in untreated samples. (D) β and γ subunits are complexed in non-ionic detergent-resistant membrane domains. IMR-32 cells were labeled for 3 h with 150 μCi [35S]methionine and lysed in TX-100–containing lysis buffer. Detergent-resistant membranes were resolubilized in octyl-glucoside containing lysis buffer. Trypsin digestion was done as described before (A–C); immunoprecipitations with ARC9 and rabbit antiβγ serum resulted in the appearance of the 27-kD and 14-kD tryptic fragments.
Mentions: We establish the occurrence of both characteristic fragments after tryptic digest of IMR-32 cell lysates using mAb ARC9 and the polyclonal rabbit anti-βγ serum (Fig. 2 A). Although we tested different concentration ranges of trypsin, we observed in these experiments the persistence of full-length forms of β, indicating that different subtype combinations of β and γ in IMR-32 cells may exist with different degrees of trypsin susceptibility, or that at least some fraction of βγ may not be fully accessible to trypsin, unlike purified βγ subunits. Re-immunoprecipitation of the 27-kD fragment with the peptide specific polyclonal antibody U-49 (derived against amino acids 131-145 of β1) established the origin of this fragment as β subunit-derived (Fig. 2 B).

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