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CXCR7 protein expression in human adult brain and differentiated neurons.

Shimizu S, Brown M, Sengupta R, Penfold ME, Meucci O - PLoS ONE (2011)

Bottom Line: Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane.Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons.Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: CXCR7 and CXCR4 are receptors for the chemokine CXCL12, which is involved in essential functions of the immune and nervous systems. Although CXCR7 transcripts are widely expressed throughout the central nervous system, little is known about its protein distribution and function in the adult brain. To evaluate its potential involvement in CXCL12/CXCR4 signaling in differentiated neurons, we studied CXCR7 protein expression in human brain and cultured neurons.

Methodology/principal findings: Immunohistochemistry and RT-PCR analyses of cortex and hippocampus from control and HIV-positive subjects provided the first evidence of CXCR7 protein expression in human adult neurons, under normal and pathological conditions. Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane. Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons. Neuronal CXCR7 co-localizes to some extent with the endoplasmic reticulum marker ERp29, but not with early/late endosome markers. Additionally, large areas of overlap are detected in the intracellular pattern of CXCR7 and CXCR4 expression.

Conclusions/significance: Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

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CXCR7 and CXCR4 protein expression in human cultured neurons.Human neurons were labeled with a plasma membrane and a nuclear marker                            prior to fixation as described previously. After permeabilization,                            neurons were stained for chemokine receptors and neuronal marker (MAP2).                            Unlike CXCR7, CXCR4 expression (12G5) in neurons was distributed to both                            the soma and processes (A) though the intracellular expression of the                            two receptors overlapped in some areas of the neuronal cell body (B).                            Negative control (NC, Mouse IgG1 isotype) is shown in lower right panel.                            Scale bars: 20 µm.
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pone-0020680-g005: CXCR7 and CXCR4 protein expression in human cultured neurons.Human neurons were labeled with a plasma membrane and a nuclear marker prior to fixation as described previously. After permeabilization, neurons were stained for chemokine receptors and neuronal marker (MAP2). Unlike CXCR7, CXCR4 expression (12G5) in neurons was distributed to both the soma and processes (A) though the intracellular expression of the two receptors overlapped in some areas of the neuronal cell body (B). Negative control (NC, Mouse IgG1 isotype) is shown in lower right panel. Scale bars: 20 µm.

Mentions: To gain greater insights about the cellular localization of CXCR7 in neurons, we studied expression of CXCR7 in human brain cultures by immunocytochemistry and confocal microscopy. Most neurons (identified by positive staining for the neuronal marker, MAP2) expressed CXCR7, mainly in perinuclear regions (Figure 4). All CXCR7-positive cells also expressed CXCR4 (Figure 4 and 5A), indicating that CXCR7 and CXCR4 were present in the same populations of neurons. However, CXCR7 was predominantly expressed in the cytoplasm, whereas CXCR4 was also found on the cell membrane. Interestingly, most CXCR7 staining overlaps with CXCR4's cytosolic staining (yellow area in Figure 5B), suggesting that the two receptors may interact at the intracellular level. The phenotype of intracellular retention of CXCR7 did not seem to be limited to neurons but also observed in some non-neuronal human cell lines (i.e. U87MG), as shown in Figure S1. This figure reports studies with different CXCR7 positive (U87MG or U343) and negative (MDA-MB-435) cells. Though CXCR7 transcripts are expressed in both U87MG and U343 cells (Figure S1A) as expected, flow cytometry and radioligand binding assays show CXCR7 protein expression only on the surface of U343 (Figure S1B). Furthermore, CXCR7 was detected in the cytoplasm of U87MG cells by confocal microscopy, but not on the cell surface (Figure S1B). Next, we asked whether this unique pattern of CXCR7 expression is human specific. To this end, we first confirmed the expression of CXCR7 mRNA in cultures of rat cortical neurons as well as in the rat cortex (Figure 6A). Then, we examined the localization of CXCR7 protein in rat neurons using different approaches. Western blot analysis showed no expression of CXCR7 in the surface protein fraction as opposed to the total protein expression (Figure 6B). Surface expression of CXCR4, which is known to internalize after CXCL12 treatment [30], was also probed using the same membrane as an internal control of protein extraction and separation. Immunostaining and confocal microscopy on rat primary neurons also confirmed the lack of CXCR7 protein expression on the neuronal surface (Figure 6C). CXCR7 staining (green) was detected predominantly in the intracellular region and did not co-localize with the membrane marker (red). To further support these results, surface expression of CXCR7 in cultured neurons was assessed by competitive binding studies. In agreement with the Western blot and confocal analysis, radioligand binding failed to demonstrate CXCR7 at the cell surface (Figure 6D/E). Rat neurons cultured for varying periods of time (i.e. up to 14 DIV) were incubated with radiolabeled CXCL12. In contrast to the control (MDA-MB-435 cells transfected with CXCR7, i.e. 435R7), minimal binding of 125I-CXCL12 is observed in rat neurons (independently of the age in vitro, data not shown), confirming the lack of surface CXCR7. Collectively, these results indicate that surface expression of CXCR7 is negligible in cultured rat/human neurons while a substantial pool of the receptor is found in the intracellular compartment.


CXCR7 protein expression in human adult brain and differentiated neurons.

Shimizu S, Brown M, Sengupta R, Penfold ME, Meucci O - PLoS ONE (2011)

CXCR7 and CXCR4 protein expression in human cultured neurons.Human neurons were labeled with a plasma membrane and a nuclear marker                            prior to fixation as described previously. After permeabilization,                            neurons were stained for chemokine receptors and neuronal marker (MAP2).                            Unlike CXCR7, CXCR4 expression (12G5) in neurons was distributed to both                            the soma and processes (A) though the intracellular expression of the                            two receptors overlapped in some areas of the neuronal cell body (B).                            Negative control (NC, Mouse IgG1 isotype) is shown in lower right panel.                            Scale bars: 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105114&req=5

pone-0020680-g005: CXCR7 and CXCR4 protein expression in human cultured neurons.Human neurons were labeled with a plasma membrane and a nuclear marker prior to fixation as described previously. After permeabilization, neurons were stained for chemokine receptors and neuronal marker (MAP2). Unlike CXCR7, CXCR4 expression (12G5) in neurons was distributed to both the soma and processes (A) though the intracellular expression of the two receptors overlapped in some areas of the neuronal cell body (B). Negative control (NC, Mouse IgG1 isotype) is shown in lower right panel. Scale bars: 20 µm.
Mentions: To gain greater insights about the cellular localization of CXCR7 in neurons, we studied expression of CXCR7 in human brain cultures by immunocytochemistry and confocal microscopy. Most neurons (identified by positive staining for the neuronal marker, MAP2) expressed CXCR7, mainly in perinuclear regions (Figure 4). All CXCR7-positive cells also expressed CXCR4 (Figure 4 and 5A), indicating that CXCR7 and CXCR4 were present in the same populations of neurons. However, CXCR7 was predominantly expressed in the cytoplasm, whereas CXCR4 was also found on the cell membrane. Interestingly, most CXCR7 staining overlaps with CXCR4's cytosolic staining (yellow area in Figure 5B), suggesting that the two receptors may interact at the intracellular level. The phenotype of intracellular retention of CXCR7 did not seem to be limited to neurons but also observed in some non-neuronal human cell lines (i.e. U87MG), as shown in Figure S1. This figure reports studies with different CXCR7 positive (U87MG or U343) and negative (MDA-MB-435) cells. Though CXCR7 transcripts are expressed in both U87MG and U343 cells (Figure S1A) as expected, flow cytometry and radioligand binding assays show CXCR7 protein expression only on the surface of U343 (Figure S1B). Furthermore, CXCR7 was detected in the cytoplasm of U87MG cells by confocal microscopy, but not on the cell surface (Figure S1B). Next, we asked whether this unique pattern of CXCR7 expression is human specific. To this end, we first confirmed the expression of CXCR7 mRNA in cultures of rat cortical neurons as well as in the rat cortex (Figure 6A). Then, we examined the localization of CXCR7 protein in rat neurons using different approaches. Western blot analysis showed no expression of CXCR7 in the surface protein fraction as opposed to the total protein expression (Figure 6B). Surface expression of CXCR4, which is known to internalize after CXCL12 treatment [30], was also probed using the same membrane as an internal control of protein extraction and separation. Immunostaining and confocal microscopy on rat primary neurons also confirmed the lack of CXCR7 protein expression on the neuronal surface (Figure 6C). CXCR7 staining (green) was detected predominantly in the intracellular region and did not co-localize with the membrane marker (red). To further support these results, surface expression of CXCR7 in cultured neurons was assessed by competitive binding studies. In agreement with the Western blot and confocal analysis, radioligand binding failed to demonstrate CXCR7 at the cell surface (Figure 6D/E). Rat neurons cultured for varying periods of time (i.e. up to 14 DIV) were incubated with radiolabeled CXCL12. In contrast to the control (MDA-MB-435 cells transfected with CXCR7, i.e. 435R7), minimal binding of 125I-CXCL12 is observed in rat neurons (independently of the age in vitro, data not shown), confirming the lack of surface CXCR7. Collectively, these results indicate that surface expression of CXCR7 is negligible in cultured rat/human neurons while a substantial pool of the receptor is found in the intracellular compartment.

Bottom Line: Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane.Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons.Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: CXCR7 and CXCR4 are receptors for the chemokine CXCL12, which is involved in essential functions of the immune and nervous systems. Although CXCR7 transcripts are widely expressed throughout the central nervous system, little is known about its protein distribution and function in the adult brain. To evaluate its potential involvement in CXCL12/CXCR4 signaling in differentiated neurons, we studied CXCR7 protein expression in human brain and cultured neurons.

Methodology/principal findings: Immunohistochemistry and RT-PCR analyses of cortex and hippocampus from control and HIV-positive subjects provided the first evidence of CXCR7 protein expression in human adult neurons, under normal and pathological conditions. Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane. Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons. Neuronal CXCR7 co-localizes to some extent with the endoplasmic reticulum marker ERp29, but not with early/late endosome markers. Additionally, large areas of overlap are detected in the intracellular pattern of CXCR7 and CXCR4 expression.

Conclusions/significance: Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

Show MeSH
Related in: MedlinePlus