Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Sep 16;286(37):32188-97.
doi: 10.1074/jbc.M111.277038. Epub 2011 Jul 5.

CXCR7/CXCR4 heterodimer constitutively recruits beta-arrestin to enhance cell migration

Fabien M Décaillot  1 , Manija A Kazmi, Ying Lin, Sarmistha Ray-Saha, Thomas P Sakmar, Pallavi Sachdev
Affiliations

CXCR7/CXCR4 heterodimer constitutively recruits beta-arrestin to enhance cell migration

Fabien M Décaillot et al. J Biol Chem. .

Abstract

G protein-coupled receptor hetero-oligomerization is emerging as an important regulator of ligand-dependent transmembrane signaling, but precisely how receptor heteromers affect receptor pharmacology remains largely unknown. In this study, we have attempted to identify the functional significance of the heteromeric complex between CXCR4 and CXCR7 chemokine receptors. We demonstrate that co-expression of CXCR7 with CXCR4 results in constitutive recruitment of β-arrestin to the CXCR4·CXCR7 complex and simultaneous impairment of G(i)-mediated signaling. CXCR7/CXCR4 co-expression also results in potentiation of CXCL12 (SDF-1)-mediated downstream β-arrestin-dependent cell signaling pathways, including ERK1/2, p38 MAPK, and SAPK as judged from the results of experiments using siRNA knockdown to deplete β-arrestin. Interestingly, CXCR7/CXCR4 co-expression enhances cell migration in response to CXCL12 stimulation. Again, inhibition of β-arrestin using either siRNA knockdown or a dominant negative mutant abrogates the enhanced CXCL12-dependent migration of CXCR4/CXCR7-expressing cells. These results show how CXCR7, which cannot signal directly through G protein-linked pathways, can nevertheless affect cellular signaling networks by forming a heteromeric complex with CXCR4. The CXCR4·CXCR7 heterodimer complex recruits β-arrestin, resulting in preferential activation of β-arrestin-linked signaling pathways over canonical G protein pathways. CXCL12-dependent signaling of CXCR4 and its role in cellular physiology, including cancer metastasis, should be evaluated in the context of potential functional hetero-oligomerization with CXCR7.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
CXCR7 alters SDF-1 signaling by forming a complex with CXCR4. A, CXCR4 and CXCR7 co-immunoprecipitate when co-expressed in HEK293 cells. Detergent-soluble lysates from HEK293 cells transfected with CXCR4-C9 and CXCR7-FLAG, as indicated, were treated with anti-FLAG antibody to immunoprecipitate (IP) CXCR7-FLAG. The pulldown was subjected to SDS-PAGE followed by Western immunoblot (IB) analysis using 1D4 mAb (upper panel). 1D4 and anti-FLAG antibodies were used to monitor CXCR4-C9 (middle panel) and CXCR7-FLAG expression levels (lower panel), respectively. B, same experiment as in A except transfected cells were preincubated with 11G8, a CXCR7-specific antibody sensitive to receptor conformation, prior to lysis. Co-IP CXCR4-C9 was detected using 1D4 antibody (upper panel), and CXCR7 levels were monitored using ant-FLAG antibody (lower panel). C, immunofluorescent staining of CXCR4 and CXCR7 expressing Neuro2A cells demonstrates co-localization of CXCR4 and CXCR7 at the membrane. D, pCRE-SEAP assay showing inhibition of FK-induced cAMP production by CXCR4 and CXCR7. SDF-1 stimulation inhibits FK-induced cellular cAMP production through CXCR4 but not through CXCR7. Co-expression of CXCR7 along with CXCR4 results in a decrease in FK-induced cellular cAMP production through CXCR4 in a dose-dependent manner. CXCR4 alone, EC50 = 3.4 ± 0.8; CXCR4 ± CXCR7 1:1.5, EC50 = 60 ± 11. E, unlike CXCR7, co-expression of CCR5 with CXCR4 does not lead to alteration in SDF-1-stimulated signaling in the pCRE-SEAP assay. CXCR4 alone, EC50 = 3.4 ± 0.8; CXCR4 ± CCR5 1:1.5, EC50 = 3.1 ± 0.5. F, pCRE-SEAP assay shows recovery of SDF-1 signaling when CXCR4-CXCR7 co-expressing cells were preincubated with 500 nm of ITAC. Data are expressed as mean ± S.E. (n = 3). CXCR4 alone, EC50 = 3.4 ± 0.8; CXCR4 ± CXCR7 1:1.5, EC50 = 60 ± 11; CXCR4 + CXCR7/ITAC, EC50 = 5.5 ± 1.7.
FIGURE 2.
FIGURE 2.
CXCR4·CXCR7 complex constitutively recruits β-arrestin. A, HEK293 cells were co-transfected with CXCR7-FLAG and β-arrestin-eGFP, along with either CXCR4 or CCR5, and treated with 100 nm SDF-1 for the indicated time. Lysates were immunoprecipitated (IP) with polyclonal anti-FLAG antibodies and subjected to Western immunoblot (IB) analysis using anti-GFP (upper panel) and anti-FLAG (middle panel) antibodies, respectively. Increased recruitment of β-arrestin to membranes containing CXCR7 is observed at base line in the presence of CXCR4 but not CCR5 (upper panel). The same trend is observed after 5 and 30 min of stimulation with SDF-1. The lower panel shows the input levels of β-arrestin-eGFP expression using anti-GFP antibody. B, quantification of β-arrestin recruitment kinetics in membranes containing CXCR7 alone or when CXCR7 is co-expressed with CXCR4 or CCR5. Data represent mean ± S.E. (n = 3). A.U., arbitrary units. C, HEK293 cells were co-transfected with CXCR7-FLAG and β-arrestin-eGFP, along with either control vector or CXCR4-C9, and treated with 100 nm SDF-1 or ITAC for 30 min. Lysates were immunoprecipitated with polyclonal anti-FLAG antibody and subjected to Western immunoblot analysis using anti-GFP antibody (bottom panel). Quantification of β-arrestin-recruitment is depicted in the top panel. Data represent results from at least three independent experiments and are expressed as mean ± S.E. The maximum response from β-arrestin recruitment to CXCR4-CXCR7 membranes is taken as 100%. Stimulation with ITAC does not result in increased recruitment of β-arrestin to the CXCR4-CXCR7-expressing membranes. D, immunofluorescence image of Neuro2A cell co-expressing CXCR4, CXCR7, and βArr-eGFP. A shows the merged image of CXCR4/CXCR7/βArr-eGFP. B–D show a higher magnification of a small area (white square, A) demonstrating co-localization of all these partners.
FIGURE 3.
FIGURE 3.
CXCR7 potentiates SDF-1-induced cell signaling pathways. A, kinetics of SDF-1-induced ERK, p38 MAPK, and SAPK/JNK activation in CXCR4-, CXCR7-, and CXCR4/CXCR7-expressing cells. HEK293 cells transfected with CXCR4 and CXCR7 alone, or in combination, were treated with 100 nm SDF-1 for 0, 5, 15, and 30 min. Cell lysates were subjected to Western immunoblot using phospho- and total ERK1/2, p38 MAPK, and SAPK/JNK antibodies. B, amount of ERK, p38 MAPK, and SAPK phosphorylation was quantified by densitometry and normalized by expressing the data as a ratio of the phosphorylated signal over the total signal. Results are expressed as a percentage of the maximum response at 5 min. Data represent mean ± S.E. (n = 3). Quantification of ERK1/2, p38 MAPK, and SAPK/JNK activation kinetics performed on samples from cells expressing CXCR4, CXCR7, or both. n.t., nontargeting.
FIGURE 4.
FIGURE 4.
β-Arrestin is required for SDF-1-induced cell signaling pathways activated by the CXCR4·CXCR7 complex. A, kinetics of SDF-1-induced ERK, p38 MAPK, and SAPK/JNK activation in cells expressing CXCR4/CXCR7 along with either nontargeting siRNA (nt siRNA) or β-arrestin2 targeting siRNA (βArr2 siRNA). Transfected cells were treated with 100 nm SDF-1 for 0, 5, 15, and 30 min. Cell lysates were subjected to Western immunoblot using phospho- and total ERK1/2, p38 MAPK, and SAPK/JNK antibodies. B, amount of ERK, p38 MAPK, and SAPK phosphorylation was quantified by densitometry and normalized by expressing the data as a ratio of the phosphorylated signal over the total signal. Results are expressed as a percentage of the maximum response at 5 min. Data represent means ± S.E. (n = 3). nt, nontargeting.
FIGURE 5.
FIGURE 5.
CXCR7 expression enhances chemotaxis. A, chemotaxis of MDA-MB-231 breast cancer cells induced by 0, 1, 10, and 100 nm of SDF-1 in the presence of increasing amounts of transfected CXCR7. Cells become more chemotactic to SDF-1 when increasing amounts of CXCR7 are expressed. B, chemotaxis of U87-CD4 cells stably expressing CXCR4 (U87-CD4-CXCR4) induced by 12.5 nm SDF-1 is enhanced in the presence of CXCR7 in a dose-dependent manner (0, 0.5, and 2 μg of CXCR7). Pretreatment of cells with 100 nm ITAC results in decreased SDF-1-stimulated migration of CXCR7 co-expressing cells. C, U87-CD4-CXCR4 cells were transfected with nontargeting (N.T.) siRNA, βArr2 siRNA, or βArr DN along with 1 μg of CXCR7. Potentiation of SDF-1-stimulated migration of U87-CD4-CXCR4 cells by CXCR7 is reverted in the presence of βArr2 siRNA and βArr DN. Data are expressed as means ± S.E. (n = 3). D, proposed model showing that in cells treated with SDF-1, CXCR4 triggers inhibition of intracellular cAMP production and Ca2+ mobilization, and both CXCR4 and CXCR7 stimulate ERK 1/2 activation (left panel). Co-expression of CXCR4 and CXCR7 leads to apparent heterodimerization of CXCR4 and CXCR7, and the heteromeric CXCR4·CXCR7 complex demonstrates a dramatically altered signaling profile. The CXCR4·CXCR7 heteromer is unable to trigger inhibition of cellular cAMP production. The constitutive recruitment of β-arrestin couples CXCR4 stimulation in the CXCR4·CXCR7 complex to proliferative pathways (ERK1/2, p38 MAPK, and SAPK).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Proudfoot A. E. (2002) Nat. Rev. Immunol. 2, 106–115 - PMC - PubMed
    1. Busillo J. M., Benovic J. L. (2007) Biochim. Biophys. Acta 1768, 952–963 - PMC - PubMed
    1. Lodowski D. T., Palczewski K. (2009) Curr. Opin. HIV AIDS 4, 88–95 - PMC - PubMed
    1. Peled A., Petit I., Kollet O., Magid M., Ponomaryov T., Byk T., Nagler A., Ben-Hur H., Many A., Shultz L., Lider O., Alon R., Zipori D., Lapidot T. (1999) Science 283, 845–848 - PubMed
    1. Alkhatib G. (2009) Curr. Opin. HIV AIDS 4, 96–103 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources