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Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma.

Brock C, Schaefer M, Reusch HP, Czupalla C, Michalke M, Spicher K, Schultz G, Nürnberg B - J. Cell Biol. (2002)

Bottom Line: This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis.Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit.Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.

ABSTRACT
Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.

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Hypothetical model for receptor- induced membrane recruitment and activation of heterodimeric PI3Kγ. fMLP receptor, a prototypical heptahelical receptor coupled to Gi proteins. PI3Kγ; the cytosolic enzyme consists of a noncatalytic p101 subunit, which is in a tight complex with p110γ, thereby stabilizing p101. The NH2 and COOH termini of p101 and p110γ are oriented in close proximity, respectively. Contact sites involve the NH2 termini of both subunits. The modular domain structure of p110γ (RBD, Ras binding domain; C2, C2 domain; hel, helical domain; N-cat, C-cat, NH2- and COOH-terminal lobes of the catalytic domain) is based on the crystal structure of an NH2-terminally truncated p110γ. (A) Membrane recruitment. The agonist-stimulated receptor induces the release of Gβγ from Gi proteins (dotted arrow). Gβγ recruits the PI3Kγ heterodimer to the plasma membrane (dotted arrow) by binding to the noncatalytic p101 (hollow arrow). Accordingly, Gβγ and p101 function as a membrane anchor and an adaptor for PI3Kγ, respectively. In addition, the C2 domain of p110γ may facilitate membrane attachment through interaction with phospholipids. p101 may also affect the interaction of PI3Kγ with the lipid interface. Membrane-attached PI3Kγ exhibits basal enzymatic activity. (B) Allosteric activation. At the membrane, Gβγ activates PI3Kγ by direct interaction with p110γ (hollow arrows). This stimulation does not require p101. However, p101 may participate in Gβγ-induced stimulation of membrane-attached p110γ. The stoichiometry of the Gβγ–PI3Kγ interaction is unknown, i.e., Gβγ may interact with p101 and p110γ through individual or common binding sites. Gβγ binds to the NH2- and COOH-terminal part of p110γ, the latter harboring the catalytic domain. Thus, Gβγ may allosterically activate PI3Kγ through a conformational change in the catalytic domain. Accordingly, Gβγ significantly increases Vmax of PtdIns-3,4,5-P3 production. PtdIns-3,4,5-P3, in turn, recruits PH domain–containing effectors such as GRP1 or Btk to the plasma membrane (dotted arrow).
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fig9: Hypothetical model for receptor- induced membrane recruitment and activation of heterodimeric PI3Kγ. fMLP receptor, a prototypical heptahelical receptor coupled to Gi proteins. PI3Kγ; the cytosolic enzyme consists of a noncatalytic p101 subunit, which is in a tight complex with p110γ, thereby stabilizing p101. The NH2 and COOH termini of p101 and p110γ are oriented in close proximity, respectively. Contact sites involve the NH2 termini of both subunits. The modular domain structure of p110γ (RBD, Ras binding domain; C2, C2 domain; hel, helical domain; N-cat, C-cat, NH2- and COOH-terminal lobes of the catalytic domain) is based on the crystal structure of an NH2-terminally truncated p110γ. (A) Membrane recruitment. The agonist-stimulated receptor induces the release of Gβγ from Gi proteins (dotted arrow). Gβγ recruits the PI3Kγ heterodimer to the plasma membrane (dotted arrow) by binding to the noncatalytic p101 (hollow arrow). Accordingly, Gβγ and p101 function as a membrane anchor and an adaptor for PI3Kγ, respectively. In addition, the C2 domain of p110γ may facilitate membrane attachment through interaction with phospholipids. p101 may also affect the interaction of PI3Kγ with the lipid interface. Membrane-attached PI3Kγ exhibits basal enzymatic activity. (B) Allosteric activation. At the membrane, Gβγ activates PI3Kγ by direct interaction with p110γ (hollow arrows). This stimulation does not require p101. However, p101 may participate in Gβγ-induced stimulation of membrane-attached p110γ. The stoichiometry of the Gβγ–PI3Kγ interaction is unknown, i.e., Gβγ may interact with p101 and p110γ through individual or common binding sites. Gβγ binds to the NH2- and COOH-terminal part of p110γ, the latter harboring the catalytic domain. Thus, Gβγ may allosterically activate PI3Kγ through a conformational change in the catalytic domain. Accordingly, Gβγ significantly increases Vmax of PtdIns-3,4,5-P3 production. PtdIns-3,4,5-P3, in turn, recruits PH domain–containing effectors such as GRP1 or Btk to the plasma membrane (dotted arrow).

Mentions: Does membrane recruitment of PI3Kγ by itself result in constitutive activation of p110γ? To answer this intriguing question, we used an isoprenylated mutant of p110γ, i.e., p110γ-CAAX, which is permanently attached to the membrane, and thereby, is in close proximity to its lipid substrates. Expression of p110γ-CAAX only slightly enhanced membrane association of fluorescent PtdIns-3,4,5-P3 sensors in HEK or VSM cells. Notably, it did not affect the morphology of HEK cells. In contrast, coexpression of Gβγ together with p110γ-CAAX induced morphological changes comparable to cells transfected with plasmids encoding Gβγ and p110γ/p101. Furthermore, stimulation of the cells with fMLP stimulated Akt phosphorylation and membrane recruitment of PH domains regardless of whether p110γ/p101 or p110γ-CAAX was the effector. This establishes a second role for Gβγ, i.e., the activation of the membrane-attached catalytic p110γ subunit even in the absence of p101 (Fig. 9). Although Gβγ interacts with either monomeric PI3Kγ subunit through individual binding sites, it does not exclude the possibility that heterodimeric PI3Kγ forms either a common or different binding site(s) for Gβγ. Attempts to determine the stoichiometry of the interaction between heterodimeric PI3Kγ and one or more Gβγ were inconclusive. One possible reason may be a weak affinity between p110γ and Gβγ, whereas p101 binds at least with moderate affinity to Gβγ as determined by copurification studies (Stephens et al., 1997; Krugmann et al., 1999; Maier et al., 1999) and by a Biacore plasmon resonance approach (unpublished data).


Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma.

Brock C, Schaefer M, Reusch HP, Czupalla C, Michalke M, Spicher K, Schultz G, Nürnberg B - J. Cell Biol. (2002)

Hypothetical model for receptor- induced membrane recruitment and activation of heterodimeric PI3Kγ. fMLP receptor, a prototypical heptahelical receptor coupled to Gi proteins. PI3Kγ; the cytosolic enzyme consists of a noncatalytic p101 subunit, which is in a tight complex with p110γ, thereby stabilizing p101. The NH2 and COOH termini of p101 and p110γ are oriented in close proximity, respectively. Contact sites involve the NH2 termini of both subunits. The modular domain structure of p110γ (RBD, Ras binding domain; C2, C2 domain; hel, helical domain; N-cat, C-cat, NH2- and COOH-terminal lobes of the catalytic domain) is based on the crystal structure of an NH2-terminally truncated p110γ. (A) Membrane recruitment. The agonist-stimulated receptor induces the release of Gβγ from Gi proteins (dotted arrow). Gβγ recruits the PI3Kγ heterodimer to the plasma membrane (dotted arrow) by binding to the noncatalytic p101 (hollow arrow). Accordingly, Gβγ and p101 function as a membrane anchor and an adaptor for PI3Kγ, respectively. In addition, the C2 domain of p110γ may facilitate membrane attachment through interaction with phospholipids. p101 may also affect the interaction of PI3Kγ with the lipid interface. Membrane-attached PI3Kγ exhibits basal enzymatic activity. (B) Allosteric activation. At the membrane, Gβγ activates PI3Kγ by direct interaction with p110γ (hollow arrows). This stimulation does not require p101. However, p101 may participate in Gβγ-induced stimulation of membrane-attached p110γ. The stoichiometry of the Gβγ–PI3Kγ interaction is unknown, i.e., Gβγ may interact with p101 and p110γ through individual or common binding sites. Gβγ binds to the NH2- and COOH-terminal part of p110γ, the latter harboring the catalytic domain. Thus, Gβγ may allosterically activate PI3Kγ through a conformational change in the catalytic domain. Accordingly, Gβγ significantly increases Vmax of PtdIns-3,4,5-P3 production. PtdIns-3,4,5-P3, in turn, recruits PH domain–containing effectors such as GRP1 or Btk to the plasma membrane (dotted arrow).
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Related In: Results  -  Collection

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fig9: Hypothetical model for receptor- induced membrane recruitment and activation of heterodimeric PI3Kγ. fMLP receptor, a prototypical heptahelical receptor coupled to Gi proteins. PI3Kγ; the cytosolic enzyme consists of a noncatalytic p101 subunit, which is in a tight complex with p110γ, thereby stabilizing p101. The NH2 and COOH termini of p101 and p110γ are oriented in close proximity, respectively. Contact sites involve the NH2 termini of both subunits. The modular domain structure of p110γ (RBD, Ras binding domain; C2, C2 domain; hel, helical domain; N-cat, C-cat, NH2- and COOH-terminal lobes of the catalytic domain) is based on the crystal structure of an NH2-terminally truncated p110γ. (A) Membrane recruitment. The agonist-stimulated receptor induces the release of Gβγ from Gi proteins (dotted arrow). Gβγ recruits the PI3Kγ heterodimer to the plasma membrane (dotted arrow) by binding to the noncatalytic p101 (hollow arrow). Accordingly, Gβγ and p101 function as a membrane anchor and an adaptor for PI3Kγ, respectively. In addition, the C2 domain of p110γ may facilitate membrane attachment through interaction with phospholipids. p101 may also affect the interaction of PI3Kγ with the lipid interface. Membrane-attached PI3Kγ exhibits basal enzymatic activity. (B) Allosteric activation. At the membrane, Gβγ activates PI3Kγ by direct interaction with p110γ (hollow arrows). This stimulation does not require p101. However, p101 may participate in Gβγ-induced stimulation of membrane-attached p110γ. The stoichiometry of the Gβγ–PI3Kγ interaction is unknown, i.e., Gβγ may interact with p101 and p110γ through individual or common binding sites. Gβγ binds to the NH2- and COOH-terminal part of p110γ, the latter harboring the catalytic domain. Thus, Gβγ may allosterically activate PI3Kγ through a conformational change in the catalytic domain. Accordingly, Gβγ significantly increases Vmax of PtdIns-3,4,5-P3 production. PtdIns-3,4,5-P3, in turn, recruits PH domain–containing effectors such as GRP1 or Btk to the plasma membrane (dotted arrow).
Mentions: Does membrane recruitment of PI3Kγ by itself result in constitutive activation of p110γ? To answer this intriguing question, we used an isoprenylated mutant of p110γ, i.e., p110γ-CAAX, which is permanently attached to the membrane, and thereby, is in close proximity to its lipid substrates. Expression of p110γ-CAAX only slightly enhanced membrane association of fluorescent PtdIns-3,4,5-P3 sensors in HEK or VSM cells. Notably, it did not affect the morphology of HEK cells. In contrast, coexpression of Gβγ together with p110γ-CAAX induced morphological changes comparable to cells transfected with plasmids encoding Gβγ and p110γ/p101. Furthermore, stimulation of the cells with fMLP stimulated Akt phosphorylation and membrane recruitment of PH domains regardless of whether p110γ/p101 or p110γ-CAAX was the effector. This establishes a second role for Gβγ, i.e., the activation of the membrane-attached catalytic p110γ subunit even in the absence of p101 (Fig. 9). Although Gβγ interacts with either monomeric PI3Kγ subunit through individual binding sites, it does not exclude the possibility that heterodimeric PI3Kγ forms either a common or different binding site(s) for Gβγ. Attempts to determine the stoichiometry of the interaction between heterodimeric PI3Kγ and one or more Gβγ were inconclusive. One possible reason may be a weak affinity between p110γ and Gβγ, whereas p101 binds at least with moderate affinity to Gβγ as determined by copurification studies (Stephens et al., 1997; Krugmann et al., 1999; Maier et al., 1999) and by a Biacore plasmon resonance approach (unpublished data).

Bottom Line: This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis.Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit.Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.

ABSTRACT
Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.

Show MeSH
Related in: MedlinePlus