The P2Y2 Nucleotide Receptor Interacts with αv Integrins to Activate Go and Induce Cell Migration

Abstract

Extracellular ATP and UTP induce chemotaxis, or directed cell migration, by stimulating the G protein-coupled P2Y 2 nucleotide receptor (P2Y 2 R). Previously, we found that an arginine-glycineaspartic acid (RGD) integrin binding domain in the P2Y 2 R enables this receptor to interact selectively with ␣ v ␤ 3 and ␣ V ␤ 5 integrins, an interaction that is prevented by mutation of the RGD sequence to arginine-glycine-glutamic acid (RGE) (Erb, L., Liu, J., Ockerhausen, J., Kong, Q., Garrad, R. C., Griffin, K., Neal, C., Krugh, B. Chemotaxis is the movement of a cell in response to a chemical gradient and is required for many physiological events, including embryonic development, immune system function, and wound healing (2-4). The process of chemotaxis is also important for understanding diseases that result from abnormal cell migration, such as chronic inflammation, atherosclerosis, and cancer metastasis. To migrate, cells must establish dynamic and highly regulated adhesive interactions with the extracellular matrix, which are mediated by integrin receptors (3). For example, recent studies have shown that ␣ v integrins play an important role in controlling cell adhesion, spreading, and motility in several cell types, including human vascular smooth muscle cells and pancreatic beta cells (5, 6). Upon activation, many types of integrin receptors cluster together and recruit a host of cytoskeletal and cytoplasmic proteins into specialized adhesive structures called focal adhesions. These focal adhesion complexes not only serve as a physical link between the extracellular and intracellular matrix but also are important sites of signal transduction for integrins and many other types of receptors that mediate cell migration (7). Chemotaxis also requires a cell to assume a polarized morphology that is controlled by cell surface receptors that activate the Rho family of GTPases, including Cdc42, Rac, and Rho (8, 9). Upon activation of a chemoattractant receptor, Cdc42 and Rac localize at the leading edge of a cell and control directional movement and the formation of lamellipodia containing highly branched actin filaments, respectively (8). Rho localizes at the rear and sides of a cell and controls the formation of contractile actin-myosin stress fibers (10). Together, these GTPases promote cell migration toward a chemoattractant by mediating extension of the actin cytoskeleton at the front edge of the cell and retraction of the cytoskeleton at the rear edge of the cell.Recent studies have shown that G protein-coupled receptors (GPCRs) 2 regulate Rac and Rac-dependent lamellipodia formation by activating the G i/o family of heterotrimeric G proteins, whereas activation of Rho and Rho-dependent stress fiber formation are controlled by the G 12/13 family (10). Furthermore, studies have shown that the ␤␥ subunits of G i/o are responsible for activation of Rac guanine nucleotide exchange factors (RacGEFs) that, in turn, activate Rac, whereas the ␣ subunits of G 12/13 are responsible for activation of RhoGEFs (8,11).The P2Y 2 ...

Concomitant activation of P2Y2 and P2Y6 receptors on monocytes is required for TLR1/2‐induced neutrophil migration by regulating IL‐8 secretion

“…Alternatively, this migration could involve chemokine-independent mechanisms such as the activation of the neutrophil P2Y 2 receptor that is instrumental for the migration of these cells caused by the Gram-positive bacteria Staphylococcus aureus in vivo [27] or expression of adhesion molecules involved in neutrophil migration, e.g. integrins [28].The results presented in this study show that a concomitant activation of TLR2 and nucleotide receptors is required for neutrophil migration both in vivo and in vitro. It is likely that nucleotides involved in this effect may be liberated from monocytes as a result of TLR2 activation but it is not excluded that this synergistic effect involves a spontaneous (basal) release of nucleotides by monocytes such as the one observed in lymphocytes.…”

“…Alternatively, this migration could involve chemokine-independent mechanisms such as the activation of the neutrophil P2Y 2 receptor that is instrumental for the migration of these cells caused by the Gram-positive bacteria Staphylococcus aureus in vivo [27] or expression of adhesion molecules involved in neutrophil migration, e.g. integrins [28].…”

Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925

“…AR-C118925 was assessed on a broad range of different targets related to the P2Y 2 receptor, including subtypes of the P2Y and P2X families, adenosine receptors, enzymes involved in nucleotide metabolism, and P2 receptor-related G protein-coupled receptors. We found that the potency of AR-C118925 is in the mid-nanomolar range on the P2Y 2 receptor and that it is at least 400-fold selective for the P2Y 2 receptor vs. its structurally and pharmacologically closest relatives, the P2Y 1 , P2Y 4 , and P2Y 6 subtypes, as well as most other P2 and adenosine receptors, and selected ectonucleotidases. Lower selectivity was observed vs. the P2Y 11 , P2X1, and P2X3 receptors (14-to 50-fold).…”

“…As a result, the second messengers inositol trisphosphate (IP 3 ) and diacylglycerol are produced that mediate the release of calcium ions from intracellular stores, and protein kinase C activation, respectively. In addition, the P2Y 2 receptor appears to couple to other G proteins as well, including G o [4], G 12 [5], and G 16 [6]. P2Y 2 receptor mRNA is expressed in a range of different human organs and tissues, including skeletal muscle and heart, at more moderate levels in the spleen, intestine, immune cells, bone marrow, and lungs, as well as to a lower extent also in kidneys, liver, stomach, pancreas, bones, and different regions of the brain [7].…”

Neuroprotective roles of the P2Y2 receptor

“…Another approach is to analyze the effects of uridine nucleotides (i.e., UTP/UDP) that only activate G qcoupled P2Y 2 , P2Y 4 , and P2Y 6 receptors among the known P2 receptor subtypes [2,4,19,20]. Among these three receptors, the P2Y 2 receptor (P2Y 2 R) has unique motifs that promote interactions with integrins and growth factor receptors, thereby enabling activation of signaling pathways beyond G q -dependent phospholipase C (PLC) [21][22][23][24]. Furthermore, the contribution of the P2Y 2 R subtype to CNS functions is becoming better understood [14,15,25] and appears to be most important under pathophysiological conditions, such as inflammation and bacterial infection [15,26].…”