Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: evidence that directed migration is mediated by betagamma dimers released by activation of Galphai-coupled receptors

Abstract

Chemotaxis is mediated by activation of seven-transmembrane domain, G protein-coupled receptors, but the signal transduction pathways leading to chemotaxis are poorly understood. To identify G proteins that signal the directed migration of cells, we stably transfected a lymphocyte cell line (300-19) with G protein-coupled receptors that couple exclusively to Galphaq (the m3 muscarinic receptor), Galphai (the kappa-opioid receptor), and Galphas (the beta-adrenergic receptor), as well as the human thrombin receptor (PAR-1) and the C-C chemokine receptor 2B. Cells expressing receptors that coupled to Galphai, but not to Galphaq or Galphas, migrated in response to a concentration gradient of the appropriate agonist. Overexpression of Galpha transducin, which binds to and inactivates free Gbetagamma dimers, completely blocked chemotaxis although having little or no effect on intracellular calcium mobilization or other measures of cell signaling. The identification of Gbetagamma dimers as a crucial intermediate in the chemotaxis signaling pathway provides further evidence that chemotaxis of mammalian cells has important similarities to polarized responses in yeast. We conclude that chemotaxis is dependent on activation of Galphai and the release of Gbetagamma dimers, and that Galphai-coupled receptors not traditionally associated with chemotaxis can mediate directed migration when they are expressed in hematopoietic cells.

Figure 1

Agonist-dependent calcium mobilization in 300-19 cells expressing G_αi- and G_αq-coupled receptors. Stably transfected 300-19 cells were incubated with or without 100 ng/ml of PTX for 24 h and loaded with Indo-1 AM. Intracellular calcium levels were measured as described. Shown are calcium fluxes in response to 100 nM of MCP-1 (CCR2B), 100 nM of U50488 (κ-opioid receptor), 200 μM of carbachol (m3 receptor), and 10 nM of thrombin (PAR-1 thrombin receptor) from a representative experiment (n = 3). The agonists used did not induce calcium fluxes in the absence of the appropriate cognate receptors.

Figure 2

Chemotaxis in 300-19 cells expressing G_αi- and G_αq-coupled receptors. Chemotaxis was analyzed by using transwells as described. Cells transfected with CCR2B and the κ-opioid receptor were incubated with or without 100 ng/ml of PTX for 24 h before the chemotaxis assays. The data are the means ± SE of four independent experiments.

Figure 3

Inhibition of chemotaxis by overexpression of G_αt. Chemotaxis was induced in cells stably expressing CCR2B or CCR2B + G_αt. Data are the means ± SE of four independent experiments.

Figure 4

Ca²⁺ mobilization in 300-19 cells expressing CCR2B and G_αt. Stably transfected 300-19 cells were loaded with Indo-1 AM, and intracellular calcium levels in response to 100 nM MCP-1 (A) or to the indicated concentrations of MCP-1 (B) were measured as described in Fig. 1. Data are representative of three independent experiments.

Figure 5

ERK activation in 300-19 cells expressing CCR2B and G_αt. Stably transfected 300-19 cells were maintained in serum-free medium for 24 h and incubated with 10 nM MCP-1 for the indicated times; lysates were assayed for ERK activation (A) as described. Quantitation is presented in B. Data are representative of three independent experiments.