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Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation.

Xiao Z, Zhang N, Murphy DB, Devreotes PN - J. Cell Biol. (1997)

Bottom Line: We found that this chimeric protein is functionally indistinguishable from wild-type cAR1.Challenge with a uniform increase in chemoattractant, sufficient to cause a dramatic decrease in the affinity of surface binding sites and cell desensitization, also did not significantly alter the distribution profile.Hence, the induced reduction in binding activity and cellular sensitivity cannot be due to receptor relocalization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.

ABSTRACT
While the localization of chemoattractant receptors on randomly oriented cells has been previously studied by immunohistochemistry, the instantaneous distribution of receptors on living cells undergoing directed migration has not been determined. To do this, we replaced cAR1, the primary cAMP receptor of Dictyostelium, with a cAR1-green fluorescence protein fusion construct. We found that this chimeric protein is functionally indistinguishable from wild-type cAR1. By time-lapse imaging of single cells, we observed that the receptors remained evenly distributed on the cell surface and all of its projections during chemotaxis involving turns and reversals of polarity directed by repositioning of a chemoattractant-filled micropipet. Thus, cell polarization cannot result from a gradient-induced asymmetric distribution of chemoattractant receptors. Some newly extended pseudopods at migration fronts showed a transient drop in fluorescence signals, suggesting that the flow of receptors into these zones may slightly lag behind the protrusion process. Challenge with a uniform increase in chemoattractant, sufficient to cause a dramatic decrease in the affinity of surface binding sites and cell desensitization, also did not significantly alter the distribution profile. Hence, the induced reduction in binding activity and cellular sensitivity cannot be due to receptor relocalization. The chimeric receptors were able to "cap" rapidly during treatment with Con A, suggesting that they are mobile in the plane of the cell membrane. This capping was not influenced by pretreatment with chemoattractant.

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cAR1-GFP is functionally indistinguishable from wild-type cAR1. (A) cAR1-GFP fusion protein as detected by immunoblotting with GFP and cAR1 antisera. Proteins were solubilized in SDS-sample buffer, resolved on 10% SDS-PAGE, and  transferred onto PVDF membranes. Duplicate samples were  loaded and analyzed in parallel. In the left lane, the fusion protein was detected with affinity-purified anti-GFP antibodies. In  the right lane, it was detected with anti-cAR1 antiserum. (B)  Both the major and minor forms of cAR1-GFP are localized to a  detergent-resistant plasma membrane subdomain. Whole cells  (lane 1) were extracted with 1.5% CHAPS at 4°C and lysate separated into soluble (lane 2) and detergent-resistant membrane  (lane 3) fractions by centrifugation, as described in Materials and  Methods. Protein samples were resolved by SDS-PAGE, transferred to membranes, and fusion protein detected by anti-cAR1  antibody. (C) Agonist-induced phosphorylation of cAR1-GFP  and gel mobility shift assay. cAR1-GFP cells were treated with  increasing doses of cAMP for 15 min to induce the phosphorylation of the COOH terminus of cAR1. cAMP doses: 1, 0 nM; 2, 5  nM; 3, 20 nM; 4, 50 nM; 5, 100 nM; 6, 200 nM; 7, 500 nM; 8, 1 μM;  and 9, 5 μM. The unmodified or phosphorylated forms of the fusion receptor were detected with cAR1 antiserum. (D) cAR1-GFP cells express the same total surface cAMP-binding sites as  wild-type cAR1 cells. cAR1-GFP and cAR1 expressing cells were  washed in PB and resuspended to 1 × 108 cells/ml density in ice-cold PB. 60 μl cells were used in the ammonium sulfate-binding  assay (Materials and Methods). The specific binding for both cell  lines in cpm were compared and later translated into the number  of binding sites per cell (see text).
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Figure 1: cAR1-GFP is functionally indistinguishable from wild-type cAR1. (A) cAR1-GFP fusion protein as detected by immunoblotting with GFP and cAR1 antisera. Proteins were solubilized in SDS-sample buffer, resolved on 10% SDS-PAGE, and transferred onto PVDF membranes. Duplicate samples were loaded and analyzed in parallel. In the left lane, the fusion protein was detected with affinity-purified anti-GFP antibodies. In the right lane, it was detected with anti-cAR1 antiserum. (B) Both the major and minor forms of cAR1-GFP are localized to a detergent-resistant plasma membrane subdomain. Whole cells (lane 1) were extracted with 1.5% CHAPS at 4°C and lysate separated into soluble (lane 2) and detergent-resistant membrane (lane 3) fractions by centrifugation, as described in Materials and Methods. Protein samples were resolved by SDS-PAGE, transferred to membranes, and fusion protein detected by anti-cAR1 antibody. (C) Agonist-induced phosphorylation of cAR1-GFP and gel mobility shift assay. cAR1-GFP cells were treated with increasing doses of cAMP for 15 min to induce the phosphorylation of the COOH terminus of cAR1. cAMP doses: 1, 0 nM; 2, 5 nM; 3, 20 nM; 4, 50 nM; 5, 100 nM; 6, 200 nM; 7, 500 nM; 8, 1 μM; and 9, 5 μM. The unmodified or phosphorylated forms of the fusion receptor were detected with cAR1 antiserum. (D) cAR1-GFP cells express the same total surface cAMP-binding sites as wild-type cAR1 cells. cAR1-GFP and cAR1 expressing cells were washed in PB and resuspended to 1 × 108 cells/ml density in ice-cold PB. 60 μl cells were used in the ammonium sulfate-binding assay (Materials and Methods). The specific binding for both cell lines in cpm were compared and later translated into the number of binding sites per cell (see text).

Mentions: We expressed the cAR1-GFP fusion construct in a cAR1- cell line, thereby replacing the endogenous receptor. Multiple lines of evidence suggested that the fusion protein correctly reported the distribution of the functionally active chemoattractant receptors. First, we investigated whether the fusion protein is effectively synthesized and processed by immunoblotting the whole cell protein extract with purified GFP antibody or cAR1 antibody (Fig. 1 A). Both antibodies detected an identical 65-kD protein, which is the expected size of the fusion protein. Smaller proteins corresponding to the size of free GFP (30 kD) or cAR1 (40 kD) were not observed. There was a faint band at 85 kD which corresponds to GFP fused to a minor 55-kD form of cAR1 routinely observed in cells expressing WT cAR1. We have speculated that this minor form may derive from a low level of premature translation from an upstream initiation site or a post-translational modification. Our previous study has established that WT cAR1 resides on special detergent-resistant microdomains of plasma membrane (Xiao and Devreotes, 1997). Crude subcellular fractionation indicated that both the major and minor GFP fusion proteins, like WT cAR1, mostly associated with this detergent-resistant subdomain (Fig. 1 B). Further fractionation of cellular lysates on sucrose density gradients showed that both bands were quantitatively localized to the plasma membrane microdomains (data not shown). All these observations indicate that cAR1-GFP is a stable fusion protein that is correctly targeted to the proper location.


Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation.

Xiao Z, Zhang N, Murphy DB, Devreotes PN - J. Cell Biol. (1997)

cAR1-GFP is functionally indistinguishable from wild-type cAR1. (A) cAR1-GFP fusion protein as detected by immunoblotting with GFP and cAR1 antisera. Proteins were solubilized in SDS-sample buffer, resolved on 10% SDS-PAGE, and  transferred onto PVDF membranes. Duplicate samples were  loaded and analyzed in parallel. In the left lane, the fusion protein was detected with affinity-purified anti-GFP antibodies. In  the right lane, it was detected with anti-cAR1 antiserum. (B)  Both the major and minor forms of cAR1-GFP are localized to a  detergent-resistant plasma membrane subdomain. Whole cells  (lane 1) were extracted with 1.5% CHAPS at 4°C and lysate separated into soluble (lane 2) and detergent-resistant membrane  (lane 3) fractions by centrifugation, as described in Materials and  Methods. Protein samples were resolved by SDS-PAGE, transferred to membranes, and fusion protein detected by anti-cAR1  antibody. (C) Agonist-induced phosphorylation of cAR1-GFP  and gel mobility shift assay. cAR1-GFP cells were treated with  increasing doses of cAMP for 15 min to induce the phosphorylation of the COOH terminus of cAR1. cAMP doses: 1, 0 nM; 2, 5  nM; 3, 20 nM; 4, 50 nM; 5, 100 nM; 6, 200 nM; 7, 500 nM; 8, 1 μM;  and 9, 5 μM. The unmodified or phosphorylated forms of the fusion receptor were detected with cAR1 antiserum. (D) cAR1-GFP cells express the same total surface cAMP-binding sites as  wild-type cAR1 cells. cAR1-GFP and cAR1 expressing cells were  washed in PB and resuspended to 1 × 108 cells/ml density in ice-cold PB. 60 μl cells were used in the ammonium sulfate-binding  assay (Materials and Methods). The specific binding for both cell  lines in cpm were compared and later translated into the number  of binding sites per cell (see text).
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Related In: Results  -  Collection

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Figure 1: cAR1-GFP is functionally indistinguishable from wild-type cAR1. (A) cAR1-GFP fusion protein as detected by immunoblotting with GFP and cAR1 antisera. Proteins were solubilized in SDS-sample buffer, resolved on 10% SDS-PAGE, and transferred onto PVDF membranes. Duplicate samples were loaded and analyzed in parallel. In the left lane, the fusion protein was detected with affinity-purified anti-GFP antibodies. In the right lane, it was detected with anti-cAR1 antiserum. (B) Both the major and minor forms of cAR1-GFP are localized to a detergent-resistant plasma membrane subdomain. Whole cells (lane 1) were extracted with 1.5% CHAPS at 4°C and lysate separated into soluble (lane 2) and detergent-resistant membrane (lane 3) fractions by centrifugation, as described in Materials and Methods. Protein samples were resolved by SDS-PAGE, transferred to membranes, and fusion protein detected by anti-cAR1 antibody. (C) Agonist-induced phosphorylation of cAR1-GFP and gel mobility shift assay. cAR1-GFP cells were treated with increasing doses of cAMP for 15 min to induce the phosphorylation of the COOH terminus of cAR1. cAMP doses: 1, 0 nM; 2, 5 nM; 3, 20 nM; 4, 50 nM; 5, 100 nM; 6, 200 nM; 7, 500 nM; 8, 1 μM; and 9, 5 μM. The unmodified or phosphorylated forms of the fusion receptor were detected with cAR1 antiserum. (D) cAR1-GFP cells express the same total surface cAMP-binding sites as wild-type cAR1 cells. cAR1-GFP and cAR1 expressing cells were washed in PB and resuspended to 1 × 108 cells/ml density in ice-cold PB. 60 μl cells were used in the ammonium sulfate-binding assay (Materials and Methods). The specific binding for both cell lines in cpm were compared and later translated into the number of binding sites per cell (see text).
Mentions: We expressed the cAR1-GFP fusion construct in a cAR1- cell line, thereby replacing the endogenous receptor. Multiple lines of evidence suggested that the fusion protein correctly reported the distribution of the functionally active chemoattractant receptors. First, we investigated whether the fusion protein is effectively synthesized and processed by immunoblotting the whole cell protein extract with purified GFP antibody or cAR1 antibody (Fig. 1 A). Both antibodies detected an identical 65-kD protein, which is the expected size of the fusion protein. Smaller proteins corresponding to the size of free GFP (30 kD) or cAR1 (40 kD) were not observed. There was a faint band at 85 kD which corresponds to GFP fused to a minor 55-kD form of cAR1 routinely observed in cells expressing WT cAR1. We have speculated that this minor form may derive from a low level of premature translation from an upstream initiation site or a post-translational modification. Our previous study has established that WT cAR1 resides on special detergent-resistant microdomains of plasma membrane (Xiao and Devreotes, 1997). Crude subcellular fractionation indicated that both the major and minor GFP fusion proteins, like WT cAR1, mostly associated with this detergent-resistant subdomain (Fig. 1 B). Further fractionation of cellular lysates on sucrose density gradients showed that both bands were quantitatively localized to the plasma membrane microdomains (data not shown). All these observations indicate that cAR1-GFP is a stable fusion protein that is correctly targeted to the proper location.

Bottom Line: We found that this chimeric protein is functionally indistinguishable from wild-type cAR1.Challenge with a uniform increase in chemoattractant, sufficient to cause a dramatic decrease in the affinity of surface binding sites and cell desensitization, also did not significantly alter the distribution profile.Hence, the induced reduction in binding activity and cellular sensitivity cannot be due to receptor relocalization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.

ABSTRACT
While the localization of chemoattractant receptors on randomly oriented cells has been previously studied by immunohistochemistry, the instantaneous distribution of receptors on living cells undergoing directed migration has not been determined. To do this, we replaced cAR1, the primary cAMP receptor of Dictyostelium, with a cAR1-green fluorescence protein fusion construct. We found that this chimeric protein is functionally indistinguishable from wild-type cAR1. By time-lapse imaging of single cells, we observed that the receptors remained evenly distributed on the cell surface and all of its projections during chemotaxis involving turns and reversals of polarity directed by repositioning of a chemoattractant-filled micropipet. Thus, cell polarization cannot result from a gradient-induced asymmetric distribution of chemoattractant receptors. Some newly extended pseudopods at migration fronts showed a transient drop in fluorescence signals, suggesting that the flow of receptors into these zones may slightly lag behind the protrusion process. Challenge with a uniform increase in chemoattractant, sufficient to cause a dramatic decrease in the affinity of surface binding sites and cell desensitization, also did not significantly alter the distribution profile. Hence, the induced reduction in binding activity and cellular sensitivity cannot be due to receptor relocalization. The chimeric receptors were able to "cap" rapidly during treatment with Con A, suggesting that they are mobile in the plane of the cell membrane. This capping was not influenced by pretreatment with chemoattractant.

Show MeSH
Related in: MedlinePlus