CysLT1 receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation

Abstract

Both cysteinyl-leukotrienes and extracellular nucleotides mediate inflammatory responses via specific G-protein-coupled receptors, the CysLT and the P2Y receptors, respectively. Since these mediators accumulate at sites of inflammation, and inflammatory cells express both classes of receptors, their responses are likely to be crossregulated. We investigated the molecular basis of desensitization and trafficking of the CysLT1 receptor constitutively and transiently expressed in the human monocyte/macrophage-like U937 or COS-7 cells in response to LTD4 or nucleotides. Exposure to agonist induced a rapid homologous desensitization of the CysLT1 receptor [as measured by the reduction in the maximal agonist-induced intracellular cytosolic Ca2+ ([Ca2+]i) transient], followed by receptor internalization (as assessed by equilibrium binding and confocal microscopy). Activation of P2Y receptors with ATP or UDP induced heterologous desensitization of the CysLT1 receptor. Conversely, LTD4-induced CysLT1 receptor activation had no effect on P2Y receptor responses, which suggests that the latter have a hierarchy in producing desensitizing signals. Furthermore, ATP/UDP-induced CysLT1 receptor desensitization was unable to cause receptor internalization, induced a faster recovery of CysLT1 functionality and was dependent upon protein kinase C. By contrast, homologous desensitization, which is probably dependent upon G-protein-receptor kinase 2 activation, induced a fast receptor downregulation and, accordingly, a slower recovery of CysLT1 functionality. Hence, CysLT1 receptor desensitization and trafficking are differentially regulated by the CysLT1 cognate ligand or by extracellular nucleotides. This crosstalk may have a profound physiological implication in the regulation of responses at sites of inflammation, and may represent just an example of a feedback mechanism used by cells to fine-tune their responses.

CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors

“…Conversely, LTD 4 -induced CysLT 1 receptor activation had no effect on P2Y receptor responses, suggesting that the latter have a hierarchy in producing desensitizing signals [14]. Thus, while a putative heterodimerization between CysLT 1 and P2Y receptors might as well explain our results, it is unlikely to occur in our system, considering the lack of cross-desensitization, the different mechanism of agonist-or nucleotides-induced CysLT 1 desensitization, and the different trafficking induced by LTD 4 or by extracellular nucleotides signals [14].…”

“…Effects on signaling at P2Y 11 , P2Y 13 and P2Y 14 receptors, although present in the dU937 cells, were not investigated. P2Y 1 receptors were included, although they are not expressed in the dU937 cells, because this is one of two P2Y receptor subtypes at which radioligand binding characterization is feasible.…”

“…Conversely, the hP2Y 1,2,4,6,11,12,13,14 receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937).…”

“…This was demonstrated in dimethyl sulfoxide-differentiated U937 (dU937) cells, an immortalized cell line known to constitutively express a high density of CysLT 1 receptors upon differentiation to monocytes/macrophages [10,11], CysLT 1 receptors respond to LTD 4 with a strong increase in cytosolic Ca 2+ concentration ([Ca 2+ ] i ) partially sensitive to PTX, and with the activation of the Ras-MAPK cascade totally dependent upon G i/o [12]. These signaling effects were totally inhibited by various specific CysLT 1 -receptor antagonists, and no CysLT 2 receptor mRNA was detected [13,14], thus indicating that in monocytemacrophage like U937 cells LTD 4 -induced responses can be totally ascribed to a CysLT 1 receptor. The promonocytic leukemia cell U937 was selected because closely related to the inflammatory cells responsible of many CysLT biological actions, and because monocyte/ macrophages activation leads to the release of a wide spectrum of cytokines and chemokines that have key roles in all inflammatory diseases.…”

“…Receptors in the A subclass (P2Y 1,2,4,6,11 ) are preferentially coupled to Gq or Gs proteins, thus mediating stimulation of phospholipase C and adenylyl cyclase. Receptors in the B subclass (P2Y 12,13,14 ) are instead mainly linked through G i activation to inhibition of adenylyl cyclase activity (ibidem).…”