Role of the intracellular domains of CXCR4 in SDF-1–mediated signaling

Abstract

The CXCR4 chemokine receptor is a G i protein-coupled receptor that triggers multiple intracellular signals in response to stromal cell-derived factor 1 (SDF-1), including calcium mobilization and p44/42 extracellular signal-regulated kinases (ERK1/2). Transduced signals lead to cell chemotaxis and are terminated through receptor internalization depending on phosphorylation of the C terminus part of CXCR4. Receptor endocytosis is also required for some receptors to stimulate ERK1/2 and to migrate through a chemokine gradient. In this study, we explored the role played by the 3 intracellular loops (ICL1-3) and the C terminus domain of CXCR4 in SDF-1-mediated signaling by using human embryonic kidney (HEK)-293 cells stably expressing wild-type or mutated forms of CXCR4. ICL3 of CXCR4 is specifically involved in G i -dependent signals such as calcium mobilization and ERK activation, but does not trigger CXCR4 internalization after SDF-1 binding, indicating that ERK phosphorylation is independent of CXCR4 endocytosis. Surprisingly, ICL2, with or without the aspartic acid, arginine, and tyrosine (DRY) motif, is dispensable for G i signaling. However, ICL2 and ICL3, as well as the C terminus part of CXCR4, are needed to transduce SDF-1-mediated chemotaxis, suggesting that this event involves multiple activation pathways and/or cooperation of several cytoplasmic domains of

On the move: endocytic trafficking in cell migration

“…Leukocytes expressing this WHIM-mutant of CXCR4 display enhanced G-proteindependent signaling and cell migration in response to CXCL12 in vitro, but are refractory to internalization, which is G-protein coupling independent [24]. However, opposing results in terms of the role of the CXCR4 C-terminus in regulating cell migration in vitro have been published [25]. A rigorous study from the Raz group demonstrated in vivo that in zebrafish CXCR4 internalization is dispensable for cell motility and directional chemokine sensing, but essential for fine-tuning of migration allowing precise arrival of primordial germ cells [26].…”

Identification of the Cytoplasmic Domains of CXCR4 Involved in Jak2 and STAT3 Phosphorylation

“…Calcium Signaling-The technique used was described previously (54). Briefly, 10 7 cells were resuspended in Hank's solution (Invitrogen, Life Technologies, Inc.), loaded with Fluo-3-AM at 2 M for 20 min at room temperature and stimulated with buffer alone or SDF-1␣ at 250 nM.…”

“…To study the direct binding of SDF-1␣ to CXCR4 expressed on transfected HEK-293 cells, 10 6 cells were incubated in 30 l of PBS for 1 h at 4°C to prevent subsequent internalization, and 30 l of a SDF-1␣ solution at 200 nM was then incubated for 20 min at 4°C after cell centrifugation. After washing with PBS, 30 l of an anti-SDF-1␣ antibody at 10 g/ml was added for 30 min, and bound Ab was revealed as described previously (54).…”

“…To address this question, we used previously constructed stable HEK-293 cells that express mutated forms of CXCR4 in which each intracellular loop (ICL) was replaced by a scrambled amino acid sequence of ICL1 that does not contain serine, threonine and tyrosine, or a CXCR4 form truncated at position 308 (54). We also constructed stable HEK-293 cells that express point mutation CXCR4 receptors in which each intracellular tyrosine, Tyr 65 (ICL1), Tyr 76 (ICL1), Tyr 135 (ICL2), or Tyr 157 (ICL2) was replaced by a nonphosphorylatable amino acid and deleted forms of ICL3.…”

Variants of CCR5, which are permissive for HIV-1 infection, show distinct functional responses to CCL3, CCL4 and CCL5

“…A single type of internalization does not explain this difference since our data suggest that the I42F and R60S variants appear to be quickly internalized, while L55Q and A73V are not. Roland et al reported that the first intracellular domain of CXCR4 was not needed to induce calcium flux or chemotaxis but did contribute to cell surface expression, 58 suggesting that the amino-terminus is linked to intracellular scaffolding maintaining receptor position. This study, in combination with our earlier study, point to a novel homologous desensitization regulatory domain comprised of the 1 and 2 TM domains and now the first intracellular loop of CCR5.…”