ATP induces intracellular calcium increases and actin cytoskeleton disaggregation via P2x receptors.

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 microM. The magnitude order for binding of different nucleotides was alpha beta-Met-ATP >or= dATPalphaS > ATP >or= ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1-100 microM) caused a concentration-dependent increase in [Ca(2+)](i)(EC(50)= 30 microM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca(2+)](e)) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca(2+) entry and increase of [Ca(2+)](i). ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca(2+)](e). Similarly, alpha,beta-Met-ATP, a potent P2x agonist also stimulated Ca(2+) mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca(2+) entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca(2+) dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.

Control of conformational equilibria in the human B2 bradykinin receptor. Modeling of nonpeptidic ligand action and comparison to the rhodopsin structure.

A prototypic study of the molecular mechanisms of activation or inactivation of peptide hormone G protein-coupled receptors was carried out on the human B2 bradykinin receptor. A detailed pharmacological analysis of receptor mutants possessing either increased constitutive activity or impaired activation or ligand recognition allowed us to propose key residues participating in intramolecular interaction networks stabilizing receptor inactive or active conformations: Asn(113) and Tyr(115) (TM III), Trp(256) and Phe(259) (TM VI), Tyr(295) (TM VII) which are homologous of the rhodopsin residues Gly(120), Glu(122), Trp(265), Tyr(268), and Lys(296), respectively. An essential experimental finding was the spatial proximity between Asn(113), which is the cornerstone of inactive conformations, and Trp(256) which plays a subtle role in controlling the balance between active and inactive conformations. Molecular modeling and mutagenesis data showed that Trp(256) and Tyr(295) constitute, together with Gln(288), receptor contact points with original nonpeptidic ligands. It provided an explanation for the ligand inverse agonist behavior on the WT receptor, with underlying restricted motions of TMs III, VI, and VII, and its agonist behavior on the Ala(113) and Phe(256) constitutively activated mutants. These data on the B2 receptor emphasize that conformational equilibria are controlled in a coordinated fashion by key residues which are located at strategic positions for several G protein-coupled receptors. They are discussed in comparison with the recently determined rhodopsin crystallographic structure.