In the brain, the P2Y11 receptor has been reported to play a role in autoimmune responses leading to narcolepsy–cataplexy (Kornum et al. 2011). This disease is characterized by loss of neurons in the hypothalamus. The single-nucleotide polymorphism (SNP) rs2305795 in the 3′ untranslated region (UTR) of the human P2RY11 receptor gene causes reduced receptor expression levels in T-lymphocytes and natural killer cells and impairs P2Y11 receptor-mediated protection against ATP-induced cell death (Kornum et al. 2011). Very little is known about functions of the P2Y11 receptor in the brain. High extracellular concentrations of ATP as a result of cell lysis promote inflammatory responses via P2Y receptor activation (Di Virgilio et al. 2009; Vitiello et al. 2012). As the P2Y11 receptor detects high extracellular ATP in the μM range (Haas et al. 2013), this receptor is predestined to playing a major role in inflammatory processes in the brain.
Another SNP in the coding region of the gene (rs3745601) results in amino acid substitution of alanine to threonine at position 87 of the P2Y11 receptor. This SNP was found to be linked to an increased risk for acute myocardial infarction (AMI) and increased levels of C-reactive protein, indicating inflammatory reactions (Black et al. 2004; Amisten et al. 2007). The association was strongest for homozygous mutations and genetically related early onset and family history AMI. AMI is often caused by atherosclerosis, which is known to be an immune-system-related disease (Tabas 2010). However, the functional and physiological impact of the A87T mutation of the human P2Y11 receptor remains unclear. We address this question in this study.
In natural killer cells which express all P2Y receptor types, ATP released by endothelial cells activates the P2Y11 receptor and inhibits chemotaxis and cytotoxicity (Gorini et al. 2010). In dendritic cells, the P2Y11 receptor regulates ATP-dependent maturation (Wilkin et al. 2001), cell migration (Schnurr et al. 2003), release of interleukin-8 (Meis et al. 2010) and interleukin-12, as well as stimulation of interleukin-10 production (Wilkin et al. 2002). The activation of the P2Y11 receptor by ATP in dendritic cells either promotes inflammation or supports immune tolerance by shaping T helper cell responses (Wilkin et al. 2002). Activation of the P2Y11 receptor with extracellular ATP inhibits the constitutive apoptosis of neutrophils (Vaughan et al. 2007). Autocrine activation of P2Y11 receptors by ATP release from intracellular vesicles causes activation of macrophages with IL-6 secretion (Sakaki et al. 2013).
In humans, the mRNA for the P2Y11 receptor is abundant in the brain, spleen, and lymphocytes, but can also be detected in macrophages, platelets, neutrophils, dendritic cells, and the heart (Berchtold et al. 1999; Moore et al. 2001; Schnurr et al. 2003; Wang et al. 2004; Wihlborg et al. 2006). The expression in platelets remains unclear as Wang and coworkers (Wang et al. 2003) found no indication of the presence of the P2Y11 receptor mRNA in platelets.
The P2Y11 receptor is a 7-transmembrane domain, G protein-coupled receptor, which belongs to the family of eight human P2Y receptors (Communi et al. 1997; Abbracchio et al. 2006). The human P2Y1 receptor is the closest homolog of the human P2Y11 receptor with 33% amino acid identity. Each P2Y receptor subtype has a distinct pattern of physiologically active adenine (P2Y1, P2Y11, P2Y12, and P2Y13 receptors) and uridine nucleotide ligands (P2Y4, P2Y6, and P2Y14 receptors). The P2Y2 receptor is activated equally well by both ATP and UTP. The receptors P2Y1 to P2Y11 are coupled to Gq signaling and therefore mediate the rise of intracellular calcium. In addition, the P2Y2 and P2Y4 receptors are linked to Gi signaling (Communi et al. 1996; Murthy and Makhlouf 1998). A unique feature within the P2Y receptor family is the P2Y11 receptor coupling to both Gq and Gs signal transduction. The latter mediates adenylyl cyclase activation and thus cAMP accumulation. The P2Y12 and P2Y13 receptors inhibit the adenylyl cyclase via Gi signaling.
The physiological standard agonist for the P2Y11 receptor is ATP, although ATP also activates P2Y1 and P2Y2 receptors. Therefore, 3′-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) is most frequently used as a potent agonist, which is selective for the P2Y11 receptor within the P2Y receptor family.
An interaction of endogenous P2Y1 receptors with P2Y11 receptors has been shown in HEK293 cells (Ecke et al. 2008). In that study, it was demonstrated that the receptor interaction results in distinct functional and pharmacological properties. Several cell types have been reported to express the P2Y1 receptor, in addition to the P2Y11 receptor. Therefore, it is important to study the consequences of an interaction between P2Y1 receptors and P2Y11 mutant receptors.
Here, we investigated the P2Y11A87T receptor in comparison with the non-mutated wild-type P2Y11 receptor to obtain insights into alterations of receptor characteristics. As P2Y11 receptor interaction with P2Y1 receptors represents a physiologically relevant situation, we used HEK293 cells to mimic this condition. We also analyzed in 1321N1 astrocytoma cells the P2Y11 receptor and its mutant. These cells lack any endogenous P2Y receptor expression. We determined the characteristics of the ligand-induced intracellular calcium responses, ATP-induced cAMP accumulation, nucleotide-induced receptor internalization, as well as the resensitization of the calcium response after a prolonged receptor desensitization period. Our results show that an impaired function of the P2Y11 receptor carrying the A87T mutation occurs only in cells that also express the P2Y1 receptor.