Abstract
Dimerization and oligomerization of G-protein coupled receptors (GPCRs) have emerged as important characters during their trans-membrane signal transduction. However, until now the relationship between GPCR dimerization and their trans-membrane signal transduction function is still uncovered. Here, using pertussis toxin (PTX) to decouple the receptor from G protein complex and with single-molecule imaging, we show that in the presence of agonist, cells treated with PTX showed a decrease in the number of dimers and oligomers on the cell surface compared with untreated ones, which suggests that oligomeric status of CXCR4 could be significantly influenced by the decoupling of G protein complex during its signal transduction process. Moreover, with chlorpromazine (CPZ) to inhibit internalization of CXCR4, it was found that after SDF-1α stimulation, cells treated with CPZ showed more dimers and oligomers on the cell surface than untreated ones, which suggest that dimers and oligomers of CXCR4 tend to internalize more easily than monomers. Taken together, our results demonstrate that dimerization and oligomerization of CXCR4 is closely related with its G protein mediated pathway and β-arrestin mediated internalization process, and would play an important role in regulating its signal transduction functions.
Highlights
G protein coupled receptors (GPCRs) form the largest and most diverse group of 7-trans-membrane proteins and play key roles in numerous processes in vivo, especially in trans-membrane signal transduction[1]
Chemotaxis experiments confirmed that after pertussis toxin (PTX) treatment the stably transfected cells lost their chemotactic activity upon SDF-1α stimulation (Fig. S1), which implied that G protein complexes have been successfully decoupled from CXCR4
Dot-blotting and confocal imaging analysis demonstrated that this PTX treatment does not affect the expression level and membrane distribution of CXCR4-EGFP (Figs S2 and S3), which is suitable for following single-molecule total internal reflection fluorescence microscopy (TIRFM) analysis
Summary
G protein coupled receptors (GPCRs) form the largest and most diverse group of 7-trans-membrane proteins and play key roles in numerous processes in vivo, especially in trans-membrane signal transduction[1]. Many GPCRs from family A and B are found existing as dimers or oligomers on the cell surface[8,12,13,14] and crystal structures were analysed[10,15] Both monomeric[16,17,18] and dimeric status of GPCRs from family A and B19 have been shown to be active enough on their own for their signal transduction function. CXCR4 belongs to family A GPCRs and is the natural receptor of stromal cell-derived growth factor (SDF-1α) It plays a key role in leukocyte trafficking, hematopoiesis, organ development and cancer metastases, as well as human immunodeficiency virus type 1(HIV-1) entry[21,22,23]. The more detailed relationship between CXCR4 dimerization, G protein complex coupling and β-arrestin mediated internalization, and whether the G protein mediated signal transduction and β-arrestin mediated internalization have an effect on the oligomeric status of the receptor, are so far unknown[6,29,30,31]
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