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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 and dissociation of GAP1IP4BP bound to PtdIns(4,5)P2 ± PtdIns(3,4,5)P3. (A) Domain organization in the primary sequence of GAP1IP4BP and its truncations. The fluorescence micrographs show HEK cells expressing the indicated constructs after 1 h stimulation with 100 nM insulin + 10% serum, 5 µM LY294002, or 10 µM wortmannin. Bar, 10 µm. (inset) The graph shows the ratio of fluorescence intensity at the plasma membrane relative to the cytosol for all cells analyzed. The fitted diffusion coefficients D and membrane dissociation times τ are shown in B and C, respectively. Results of statistical analysis are shown in Tables II and III. Data are means ± SEM.
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fig7: Diffusion and dissociation of GAP1IP4BP bound to PtdIns(4,5)P2 ± PtdIns(3,4,5)P3. (A) Domain organization in the primary sequence of GAP1IP4BP and its truncations. The fluorescence micrographs show HEK cells expressing the indicated constructs after 1 h stimulation with 100 nM insulin + 10% serum, 5 µM LY294002, or 10 µM wortmannin. Bar, 10 µm. (inset) The graph shows the ratio of fluorescence intensity at the plasma membrane relative to the cytosol for all cells analyzed. The fitted diffusion coefficients D and membrane dissociation times τ are shown in B and C, respectively. Results of statistical analysis are shown in Tables II and III. Data are means ± SEM.

Mentions: The highly homologous GAP1IP4BP makes an interesting contrast with GAP1m because in addition to binding PtdIns(3,4,5)P3, GAP1IP4BP protein also binds with high affinity to PtdIns(4,5)P2 (Cozier et al., 2000). As a result, this protein is constitutively targeted to the plasma membrane (Fig. 7 A). Under conditions of no PtdIns(3,4,5)P3 production, there is a slight cytosolic haze of unbound protein (Fig. 7 A), but the majority is bound to the plasma membrane, where it binds with an apparent τ of ∼3.5 s and moves with a lateral diffusion coefficient somewhat slower than GAP1m at ∼0.3 µm2/s (see also Brough et al., 2005). Activation of PtdIns(3,4,5)P3 synthesis in the cells leads to a minor effect on the lateral diffusion coefficient (Fig. 7 B and Table II). However, it causes an increase in the ratio of plasma membrane to cytosolic fluorescence (Fig. 7 A), as the cytosolic haze is no longer discernable in most cells. This was accompanied by a nearly twofold increase in the dissociation time (Fig. 7 C). This result was unexpected because even at the height of PI 3-kinase activation, plasma membrane PtdIns(3,4,5)P3 levels reach only a fraction (<10%) of PtdIns(4,5)P2 levels (Stephens et al., 1993), but it does suggest that GAP1IP4BP can recognize receptor-generated PtdIns(3,4,5)P3, and this in turn points to the possibility that it might be a PtdIns(3,4,5)P3 effector.


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 and dissociation of GAP1IP4BP bound to PtdIns(4,5)P2 ± PtdIns(3,4,5)P3. (A) Domain organization in the primary sequence of GAP1IP4BP and its truncations. The fluorescence micrographs show HEK cells expressing the indicated constructs after 1 h stimulation with 100 nM insulin + 10% serum, 5 µM LY294002, or 10 µM wortmannin. Bar, 10 µm. (inset) The graph shows the ratio of fluorescence intensity at the plasma membrane relative to the cytosol for all cells analyzed. The fitted diffusion coefficients D and membrane dissociation times τ are shown in B and C, respectively. Results of statistical analysis are shown in Tables II and III. Data are means ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Diffusion and dissociation of GAP1IP4BP bound to PtdIns(4,5)P2 ± PtdIns(3,4,5)P3. (A) Domain organization in the primary sequence of GAP1IP4BP and its truncations. The fluorescence micrographs show HEK cells expressing the indicated constructs after 1 h stimulation with 100 nM insulin + 10% serum, 5 µM LY294002, or 10 µM wortmannin. Bar, 10 µm. (inset) The graph shows the ratio of fluorescence intensity at the plasma membrane relative to the cytosol for all cells analyzed. The fitted diffusion coefficients D and membrane dissociation times τ are shown in B and C, respectively. Results of statistical analysis are shown in Tables II and III. Data are means ± SEM.
Mentions: The highly homologous GAP1IP4BP makes an interesting contrast with GAP1m because in addition to binding PtdIns(3,4,5)P3, GAP1IP4BP protein also binds with high affinity to PtdIns(4,5)P2 (Cozier et al., 2000). As a result, this protein is constitutively targeted to the plasma membrane (Fig. 7 A). Under conditions of no PtdIns(3,4,5)P3 production, there is a slight cytosolic haze of unbound protein (Fig. 7 A), but the majority is bound to the plasma membrane, where it binds with an apparent τ of ∼3.5 s and moves with a lateral diffusion coefficient somewhat slower than GAP1m at ∼0.3 µm2/s (see also Brough et al., 2005). Activation of PtdIns(3,4,5)P3 synthesis in the cells leads to a minor effect on the lateral diffusion coefficient (Fig. 7 B and Table II). However, it causes an increase in the ratio of plasma membrane to cytosolic fluorescence (Fig. 7 A), as the cytosolic haze is no longer discernable in most cells. This was accompanied by a nearly twofold increase in the dissociation time (Fig. 7 C). This result was unexpected because even at the height of PI 3-kinase activation, plasma membrane PtdIns(3,4,5)P3 levels reach only a fraction (<10%) of PtdIns(4,5)P2 levels (Stephens et al., 1993), but it does suggest that GAP1IP4BP can recognize receptor-generated PtdIns(3,4,5)P3, and this in turn points to the possibility that it might be a PtdIns(3,4,5)P3 effector.

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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Related in: MedlinePlus