A truncated naturally occurring variant of the human receptor P2X 7 was identified in cancer cervical cells. The novel protein (P2X 7-j ), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X 7 receptor. The P2X 7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X 7 receptor agonist benzoyl-ATP. The P2X 7-j interacted with the full-length P2X 7 in a manner suggesting heterooligomerization and blocked the P2X 7 -mediated actions. Interestingly, P2X 7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but fulllength P2X 7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X 7 immunoreactivity suggesting lack of P2X 7 homo(tri)-oligomerization. These results identify a novel P2X 7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.The receptor P2X 7 belongs to the P2X subfamily of P2 nucleotide receptors (1, 2), which are membrane-bound, ligand-operated channels (3-5). ATP is the naturally occurring ligand for the P2X 7 and activation of the receptor by brief exposure to extracellular ATP opens cation channels that allow Ca 2ϩ , Na ϩ , and K ϩ influx (6). Longer exposure to ATP allows passage of cations with progressively larger diameters, up to 900 Da, through formation of pores (7). The mechanism of pore formation is unclear, and opinions vary between decreased filter selectivity of existing channels (8) to rearrangement of receptor molecules (9). P2X 7 receptors function in a cell-specific manner and effects of receptor activation are determined by receptor expression (10), trafficking and plasma membrane localization (11-13), oligomerization (5), and postactivation internalization, recycling, and degradation (14). Expression of P2X 7 can be regulated hormonally; in human cervical epithelial cells epinephrine down-regulates expression of the glycosylated form of the P2X 7 and increases receptor degradation, and the effects can be potentiated by epidermal growth factor (15). Evidence for the physiological role of the P2X 7 comes from studies of P2X 7 -deficient mice, indicating its role in inflammatory (16) and immune processes (17). Epithelial cells of the female lower reproductive tract express the P2X 7 (18), and in human cervical epithelial cells ligand binding induces apoptosis by a mechanism that involves pore formation, augmented calcium influx, and calcium-dependent activation of the apoptotic mitochondrial pathway (19,20). Because human cervical epithelial cells secrete ATP into the extra...

In the current model of receptor activation, the given hormone is not involved in the conversion of the inactive receptor (R) to the fully active state (R*). Rather, it preferentially selects the activated receptor conformation, thereby shifting the equilibrium toward R*. The hormone angiotensin II (Ang II) contains two residues, Tyr4 and Phe8, that are essential for agonism. We show that the conserved Asn111 in transmembrane helix III of the AT1 angiotensin receptor directly interacts with the Tyr4 side chain. A decrease in the size of the Asn111 side chain induces an intermediate activated receptor conformation (R'). The Ang II analogue [Sar1,Ile4,Ile8]Ang II fully activates the N111G mutant, indicating that either the transition from R' to R* or the stabilization of the R* state requires binding by Ang II but not its Tyr4 and Phe8 side chains. In contrast, [Sar1,Ile4,Ile8]Ang II binds to but does not activate the wild-type AT1 receptor (R), suggesting that in the wild-type receptor spontaneous occurrence of R' and R* states is rare. Thus, Ang II through interactions involving Tyr4 and Phe8 induces a transition from R to R' and through unspecified interactions induces transition from R' to R* states rather than stabilizing the spontaneously generated R* state by "conformational, selection".

Normal human ectocervical epithelial (hECE) cells undergo apoptosis in culture. Baseline apoptosis could be increased by shifting cells to serum-free medium and blocked by lowering extracellular calcium. Treatment with the ATPase apyrase attenuated baseline apoptosis, suggesting that extracellular ATP and purinergic mechanisms control the apoptosis. Treatment with ATP and the P2X7 receptor analog 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased apoptosis significantly, in a time- and dose-related manner. The threshold of ATP effect was 0.5 microM in hECE cells and approximately 1 microM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-alpha, and it could be attenuated by lowering extracellular calcium and by treatment with the caspase-9 inhibitor Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK). Treatment with BzATP activated caspase-9, and, in contrast to TNF-alpha, it had only a mild effect on caspase-8. Both BzATP and TNF-alpha activated caspase-3, suggesting that BzATP activates predominantly the mitochondrial apoptotic pathway. Both hECE and CaSki cells secrete ATP into the extracellular fluid, and mean ATP activity in conditioned medium was approximately 0.5 microM, which is in the range of values that suffice to activate the P2X7 receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X7 receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway.

The objective of this study was to understand the mechanisms involved in P2X 7 receptor activation. Treatments with ATP or with the P2X 7 receptor-specific ligand 2′,3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP) induced pore formation, but the effect was slower in CaSki cells expressing endogenous P2X 7 receptor than in human embryonic kidney (HEK)-293 cells expressing exogenous P2X 7 receptor (HEK-293-hP2X 7 -R). In both types of cells Western blots revealed expression of three forms of the receptor: the functional 85-kDa form present mainly in the membrane and 65-and 18-kDa forms expressed in both the plasma membrane and the cytosol. Treatments with ATP transiently decreased the 85-kDa form and increased the 18-kDa form in the membrane, suggesting internalization, degradation, and recycling of the receptor. In CaSki cells ATP stimulated phosphorylation of the 85-kDa form on tyrosine and serine residues. Phosphorylation on threonine residues increased with added ATP, and it increased ATP requirements for phosphorylation on tyrosine and serine residues, suggesting a dominant-negative effect. In both CaSki and in HEK-293-hP2X 7 -R cells ATP also increased binding of the 85-kDa form to G protein-coupled receptor kinase (GRK)-3, β-arrestin-2, and dynamin, and it stimulated β-arrestin-2 redistribution into submembranous regions of the cell. These results suggest a novel mechanism for P2X 7 receptor action, whereby activation involves a GRK-3-, β-arrestin-2-, and dynamin-dependent internalization of the receptor into clathrin domains, followed in part by receptor degradation as well as receptor recycling into the plasma membrane.Keywords purinergic receptor; recycling; dynamin; clathrin; cervix; epithelium The P2X 7 Receptor Belongs to the P2X receptor subfamily of P2 nucleotide receptors (6,45), which are membrane-bound, ligand-operated K + -, Na + -, and Ca 2+ -permeable channels that function as homo-or heteromultimeric complexes (38,48). Activation of the P2X 7 receptor can stimulate various cell-specific signaling cascades (11,40). Effects unique to the receptor are the induction of membrane fusion and blebbing associated with microvesicle generation (9,10), interleukin-1β processing and secretion (11,24,33), and opening of membrane pores (11,37,40,46). Epithelial cells of the female lower reproductive tract express the P2X 7 receptor Address for reprint requests and other correspondence: G. I. Gorodeski, University MacDonald Women's Hospital, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106 (E-mail: gig@cwru.edu). NIH Public AccessAuthor Manuscript Am J Physiol Cell Physiol. Author manuscript; available in PMC 2008 December 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (3), and in human cervical epithelial cells activation of the receptor induces apoptosis by a mechanism that involves calcium-dependent activation of the mitochondrial pathway (51). Apoptosis plays an important role in the regulation of cell cycle and control of neoplas...

The AT1 receptor is a G-protein-coupled receptor (GPCR); its activation from the basal state (R) requires an interaction between Asn111 in transmembrane helix III (TM-III) of the receptor and the Tyr4 residue of angiotensin II (Ang II). Asn111 to Gly111 mutation (N111G) results in constitutive activation of the AT1 receptor (Noda et al. (1996) Biochemistry, 35, 16435-16442). We show here that replacement of the AT1 receptors TM-III with a topologically identical 16-residue segment (Cys101-Val116) from the AT2 receptor induces constitutive activity, although Asn111 is preserved in the resulting chimera, CR18. Effects of CR18 and N111G mutations are neither additive nor synergistic. The conformation(s) induced in either mutant mimics the partially activated state (R'), and transition to the fully activated R conformation in both no longer requires the Tyr4 of Ang II. Both the R state of the receptor and the Tyr4 Ang II dependence of receptor activation can be reinstated by introduction of a larger sized Phe side chain at the 111 position in CR18, suggesting that the CR18 mutation generated an effect similar to the reduction of side chain size in the N111G mutation. Consistently in the native AT1 receptor, R' conformation is generated by replacement with residues smaller but not larger than the Asn111. However, size substitution of several other TM-III residues in both receptors did not affect transitions between R, R', and R states. Thus, the property responsible for Asn111 function as a conformational switch is neither polarity nor hydrogen bonding potential but the side chain size. We conclude that the fundamental mechanism responsible for constitutive activation of the AT1 receptor is to increase the entropy of the key agonist-switch binding residue, Asn111. As a result, the normally agonist-dependent R --> R' transition occurs spontaneously. This mechanism may be applicable to many other GPCRs.