Abstract
In the human neoplastic cell lines 5637 and HeLa, recombinant CXCL12 elicited, as expected, downstream signals via both G-protein-dependent and β-arrestin-dependent pathways responsible for inducing a rapid and a late wave, respectively, of ERK1/2 phosphorylation. In contrast, the structural variant [N33A]CXCL12 triggered no β-arrestin-dependent phosphorylation of ERK1/2, and signaled via G protein-dependent pathways alone. Both CXCL12 and [N33A]CXCL12, however, generated signals that transinhibited HER1 phosphorylation via intracellular pathways. 1) Prestimulation of CXCR4/HER1-positive 5637 or HeLa cells with CXCL12 modified the HB-EGF-dependent activation of HER1 by delaying the peak phosphorylation of tyrosine 1068 or 1173. 2) Prestimulation with the synthetic variant [N33A]CXCL12, while preserving CXCR4-related chemotaxis and CXCR4 internalization, abolished HER1 phosphorylation. 3) In cells knockdown of β-arrestin 2, CXCL12 induced a full inhibition of HER1 like [N33A]CXCL12 in non-silenced cells. 4) HER1 phosphorylation was restored as usual by inhibiting PCK, calmodulin or calcineurin, whereas the inhibition of CaMKII had no discernable effect. We conclude that both recombinant CXCL12 and its structural variant [N33A]CXCL12 may transinhibit HER1 via G-proteins/calmodulin/calcineurin, but [N33A]CXCL12 does not activate β-arrestin-dependent ERK1/2 phosphorylation and retains a stronger inhibitory effect. Therefore, we demonstrated that CXCL12 may influence the magnitude and the persistence of signaling downstream of HER1 in turn involved in the proliferative potential of numerous epithelial cancer. In addition, we recognized that [N33A]CXCL12 activates preferentially G-protein-dependent pathways and is an inhibitor of HER1.
Highlights
CXCL12 regulates important hematopoietic functions, induces cell adhesion and chemotaxis, and coordinates the circulation of hematopoietic stem cells, lymphocytes and monocytes [1], [2], [3]
Because human EGF receptor 1 (HER1) transactivation via binding of a shedded ligand would be indicated by HER1 internalization, we concluded that CXCL12 did not induce HER1 transactivation in 5637 or HeLa cells through this mechanism
The current study in 5637 and HeLa cells defines a novel mechanism of crosstalk between CXCR4 and HER1, whereby stimulation with recombinant CXCL12 modifies the pattern of HER1 phosphorylation via G-proteins/calmodulin/calcineurin
Summary
CXCL12 regulates important hematopoietic functions, induces cell adhesion and chemotaxis, and coordinates the circulation of hematopoietic stem cells, lymphocytes and monocytes [1], [2], [3]. Though the role of CXCR7 is not fully understood up to date [10], [11], it is known that downstream of CXCR4 native CXCL12 activates both G-protein-dependent and b-arrestin-dependent signaling pathways, which may support distinct intracellular messages [12], [13], [14]. It seems that structural variants of either the ligand or the receptor may result in the dissociated activation of either G-protein- or b-arrestindependent pathways, followed by different cellular responses [13], [15], [18]
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