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Reversible binding and rapid diffusion of proteins in complex with inositol lipids serves to coordinate free movement with spatial information.

Hammond GR, Sim Y, Lagnado L, Irvine RF - J. Cell Biol. (2009)

Bottom Line: However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions.We find that the protein-lipid complexes retain a relatively rapid ( approximately 0.1-1 microm(2)/s) diffusion coefficient in the membrane, likely dominated by protein-protein interactions, but the limited time scale (seconds) of these complexes, dictated principally by lipid-protein interactions, limits their range of action to a few microns.Moreover, our data reveal that GAP1(IP4BP), a protein that binds PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) in vitro with similar affinity, is able to "read" PtdIns(3,4,5)P(3) signals in terms of an elongated residence time at the membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, England, UK. gruh2@cam.ac.uk

ABSTRACT
Polyphosphoinositol lipids convey spatial information partly by their interactions with cellular proteins within defined domains. However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions. To investigate quantitatively how these conflicting functional needs are optimized, we used different fluorescence recovery after photobleaching techniques to investigate inositol lipid-effector protein kinetics in terms of the real-time dissociation from, and diffusion within, the plasma membrane. We find that the protein-lipid complexes retain a relatively rapid ( approximately 0.1-1 microm(2)/s) diffusion coefficient in the membrane, likely dominated by protein-protein interactions, but the limited time scale (seconds) of these complexes, dictated principally by lipid-protein interactions, limits their range of action to a few microns. Moreover, our data reveal that GAP1(IP4BP), a protein that binds PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) in vitro with similar affinity, is able to "read" PtdIns(3,4,5)P(3) signals in terms of an elongated residence time at the membrane.

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Diffusion of GAP1m in the presence and absence of PtdIns(3,4,5)P3. (A) Fluorescence micrographs of HEK cells expressing GFP-GAP1m after 1 h stimulation with 100 nM insulin in 10% serum, or 5 µM LY294002 in serum-free medium as indicated. Bar, 10 µm. (B) Diffusion coefficients under the above conditions; cytosolic diffusion in the presence of LY294002 is significantly faster than on the membrane in the presence of insulin (P < 0.0001, Mann-Whitney test). Data are means ± SEM.
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fig6: Diffusion of GAP1m in the presence and absence of PtdIns(3,4,5)P3. (A) Fluorescence micrographs of HEK cells expressing GFP-GAP1m after 1 h stimulation with 100 nM insulin in 10% serum, or 5 µM LY294002 in serum-free medium as indicated. Bar, 10 µm. (B) Diffusion coefficients under the above conditions; cytosolic diffusion in the presence of LY294002 is significantly faster than on the membrane in the presence of insulin (P < 0.0001, Mann-Whitney test). Data are means ± SEM.

Mentions: GAP1m contains a PH domain with high affinity for PtdIns(3,4,5)P3, and so translocates to the plasma membrane in response to PI 3-kinase activation (Lockyer et al., 1999). HEK cells transfected with GFP-GAP1m and incubated in serum-free medium and a PI 3-kinase inhibitor (to ensure no production of PtdIns(3,4,5)P3) showed cytosolic fluorescence (Fig. 6 A) and a rapid diffusion coefficient of ∼4 µm2/s (Fig. 6 B and Table I). Conversely, addition of insulin to the cells in serum-containing medium induces PtdIns(3,4,5)P3 synthesis and partial translocation of the protein to the plasma membrane (Fig. 6 A), where, like PH-PLCδ1, it exhibits a slower D of around ∼1.3 µm2/s (Fig. 6 B) and a τ of ∼3.4 s (Table I). This confirms that when bound to membranes, GFP-GAP1m retains a high mobility (Brough et al., 2005), though that earlier study overestimated the D by not taking dissociation into account, thus missing the detectable decrease in D value caused by membrane binding (Table I).


Reversible binding and rapid diffusion of proteins in complex with inositol lipids serves to coordinate free movement with spatial information.

Hammond GR, Sim Y, Lagnado L, Irvine RF - J. Cell Biol. (2009)

Diffusion of GAP1m in the presence and absence of PtdIns(3,4,5)P3. (A) Fluorescence micrographs of HEK cells expressing GFP-GAP1m after 1 h stimulation with 100 nM insulin in 10% serum, or 5 µM LY294002 in serum-free medium as indicated. Bar, 10 µm. (B) Diffusion coefficients under the above conditions; cytosolic diffusion in the presence of LY294002 is significantly faster than on the membrane in the presence of insulin (P < 0.0001, Mann-Whitney test). Data are means ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654307&req=5

fig6: Diffusion of GAP1m in the presence and absence of PtdIns(3,4,5)P3. (A) Fluorescence micrographs of HEK cells expressing GFP-GAP1m after 1 h stimulation with 100 nM insulin in 10% serum, or 5 µM LY294002 in serum-free medium as indicated. Bar, 10 µm. (B) Diffusion coefficients under the above conditions; cytosolic diffusion in the presence of LY294002 is significantly faster than on the membrane in the presence of insulin (P < 0.0001, Mann-Whitney test). Data are means ± SEM.
Mentions: GAP1m contains a PH domain with high affinity for PtdIns(3,4,5)P3, and so translocates to the plasma membrane in response to PI 3-kinase activation (Lockyer et al., 1999). HEK cells transfected with GFP-GAP1m and incubated in serum-free medium and a PI 3-kinase inhibitor (to ensure no production of PtdIns(3,4,5)P3) showed cytosolic fluorescence (Fig. 6 A) and a rapid diffusion coefficient of ∼4 µm2/s (Fig. 6 B and Table I). Conversely, addition of insulin to the cells in serum-containing medium induces PtdIns(3,4,5)P3 synthesis and partial translocation of the protein to the plasma membrane (Fig. 6 A), where, like PH-PLCδ1, it exhibits a slower D of around ∼1.3 µm2/s (Fig. 6 B) and a τ of ∼3.4 s (Table I). This confirms that when bound to membranes, GFP-GAP1m retains a high mobility (Brough et al., 2005), though that earlier study overestimated the D by not taking dissociation into account, thus missing the detectable decrease in D value caused by membrane binding (Table I).

Bottom Line: However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions.We find that the protein-lipid complexes retain a relatively rapid ( approximately 0.1-1 microm(2)/s) diffusion coefficient in the membrane, likely dominated by protein-protein interactions, but the limited time scale (seconds) of these complexes, dictated principally by lipid-protein interactions, limits their range of action to a few microns.Moreover, our data reveal that GAP1(IP4BP), a protein that binds PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) in vitro with similar affinity, is able to "read" PtdIns(3,4,5)P(3) signals in terms of an elongated residence time at the membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, England, UK. gruh2@cam.ac.uk

ABSTRACT
Polyphosphoinositol lipids convey spatial information partly by their interactions with cellular proteins within defined domains. However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions. To investigate quantitatively how these conflicting functional needs are optimized, we used different fluorescence recovery after photobleaching techniques to investigate inositol lipid-effector protein kinetics in terms of the real-time dissociation from, and diffusion within, the plasma membrane. We find that the protein-lipid complexes retain a relatively rapid ( approximately 0.1-1 microm(2)/s) diffusion coefficient in the membrane, likely dominated by protein-protein interactions, but the limited time scale (seconds) of these complexes, dictated principally by lipid-protein interactions, limits their range of action to a few microns. Moreover, our data reveal that GAP1(IP4BP), a protein that binds PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) in vitro with similar affinity, is able to "read" PtdIns(3,4,5)P(3) signals in terms of an elongated residence time at the membrane.

Show MeSH
Related in: MedlinePlus