The carboxyl terminus (C-tail) of G protein-coupled receptors is divergent in length and structure and may represent an individualized cytoplasmic domain. By progressively truncating the A 1 adenosine receptor, a G i/o -coupled receptor with short cytoplasmic stretches, we identify two inherent functions of the C-tail, namely a role in receptor export from the endoplasmic reticulum (ER) and a role in G protein coupling. Deletion of the last 22 and 26 amino acids (of 36) reduced and completely abolished surface expression of the receptor, respectively. The severely truncated receptors were retained in the ER and failed to bind ligands. If overexpressed, even a substantial portion of the fulllength receptor was retained in the ER in a form that was not functional. These data indicate that folding is rate limiting in export from the ER and that the proximal segment of the carboxyl terminus provides a docking site for the machinery involved in folding and quality control. In addition, the proximal portion is also important in G protein coupling. This latter role was unmasked when the distal portion of the C-tail (the extreme 18 amino acids, including a palmitoylated cysteine) had been removed; the resulting receptor was functional and transferred the agonist-mediated signal more efficiently than the full-length receptor. Signaling was enhanced because the coupling affinity increased (by 3-fold), which translated into a higher agonist potency. Thus, the distal portion of the carboxyl terminus provides for an autoinhibitory restraint, presumably by folding back and preventing G protein access to the proximal part of the C-tail.The cytoplasmic face of a G protein-coupled receptor (GPCR) executes the signal transfer reaction through interaction with G proteins and accessory non-GTP-binding protein components. The interface is composed of three intracellular loops that connect the trans-membrane helices and a fourth loop created by a segment of 8 -16 amino acids adjacent to transmembrane helix 7 (TM7), which is tethered to the phospholipid bilayer by one or two palmitoylated cysteine residues. In individual instances, each of the intracellular segments has been shown to contribute to G protein coupling and signaling (1). The second intracellular loop (i 2 ), which contains the conserved D(E)RY(W) motif and the third intracellular loop (i 3 ), in particular its amino-and carboxyl-terminal ends, are key elements. Between receptor types and even among related subtypes, sequence similarities in the cytoplasmic domains are scarce; this variability suggests that, among the cytoplasmic segments, some are reserved for receptor functions more divergent than G protein coupling.The receptor carboxyl terminus varies considerably in length ranging from nil (in the GnRH receptor) to more than 200 amino acids (in the Ca 2ϩ /Mg 2ϩ -sensing receptor). Distinct functional properties have been assigned to individual carboxyl termini. In rhodopsin and the metabotropic glutamate receptor (subtypes IV, VI, and VII), the proximal portion repre...

Although feedback inhibition of noradrenaline release by coreleased nucleotides is a well known phenomenon, it remained unclear which P2 receptor subtypes and associated signalling cascades may be involved. In the rat pheochromocytoma cell line PC12, 2-methylthio-ADP reduced noradrenaline release triggered by K+ depolarization more potently than ADP and ATP, whereas UDP or UTP failed to do so. The inhibition by ADP was abolished by pertussis toxin and antagonized by reactive blue 2, 2-methylthio-AMP, and AR-C69931MX, but not by suramin. AR-C69931MX acted as a competitive antagonist with an apparent affinity of 2 nm, but did not alter noradrenaline release, when PC12 cells were continuously superfused. However, when the superfusion was halted during K+ depolarization, release was significantly reduced and this inhibition was attenuated by AR-C69931MX, thus revealing ongoing autoinhibition. Rises in cellular cyclic AMP did not alter depolarization-evoked release nor its reduction by ADP, even though the nucleotide did inhibit cyclic AMP accumulation. ADP and the direct Ca2+ channel blocker Cd2+ inhibited voltage-activated Ca2+ currents, but not ATP-induced currents, and both agents reduced K+-evoked, but not ATP-evoked, release. Hence, if voltage-gated Ca2+ channels do not contribute to stimulation-evoked release, ADP fails to exert its inhibitory action. In primary cultures of rat sympathetic neurons, ADP also reduced Ca2+ currents and K+-evoked noradrenaline release, and AR-C69931MX acted again as competitive antagonist with an apparent affinity of 3 nm. These results show that P2Y12 receptors mediate an autoinhibition of transmitter release from PC12 cells and sympathetic neurons through an inhibition of voltage-gated Ca2+ channels.

Exchanging each of the conserved aromatic residues of the NPxxY(x) 5,6 F sequence (at the boundary of helices 7 and 8) generated variants of the A 1 adenosine receptor that were retained within the cell. The mutations disconnected a link between ␣-helix 7 and cytosolic helix 8, likely destabilizing the structure of the proximal carboxyl terminus. The mutant receptors were rescued by incubation of cells with a pharmacochaperone, a membrane-permeable ligand that homosterically binds to the receptor; pharmacochaperoning restored the density of functional receptors at the plasma membrane. The following observations support the assumption that retention and the site of pharmacochaperone action were within bounds of the endoplasmic reticulum (ER): 1) the retained receptor colocalized with an ER marker; 2) pharmacochaperoning initiated receptor transfer to Golgi stacks; and 3) the inhibitor of glycoprotein synthesis tunicamycin suppressed receptor chaperoning. Our data are consistent with the hypothesis that pharmacochaperoning stabilizes the structure of late folding intermediates and lifts a block on maturation, allowing the receptors to exit from the ER. We suggest that the ER-associated 40-kDa heat shock protein family member D 1 receptor interacting protein 78 (DRiP78; M r , ϳ78,000) represents a model executor of quality control. Overexpressed DRiP78 interacted physically with the A 1 receptor, inhibited export to the plasma membrane, and in this action was selective for the mutants relative to the wild-type receptor. Both agonist and antagonist were effective chaperone ligands. Thus, occupancy of the binding pocket corrected the mutation-induced disorder, indicating a mutual impingement of the transmembrane domain and the proximal carboxyl terminus in establishing the stable receptor fold.G protein-coupled receptors are glycosylated integral membrane proteins localized at the cell surface. At any time, their density on the cell surface is the result of receptor trafficking to and sequestration away from the plasma membrane. Delivery to the cell surface is caused by recycling of internalized receptors, or it follows export of newly synthesized receptors. Up-regulation of functional receptors caused by receptorspecific ligands may be caused by diminished internalization or accelerated export. The concept of pharmacochaperoning refers to the latter (Morello et al., 2000).Chaperone ligands (pharmacochaperones) have been found to deliver retained receptors to the cell surface and afford functional rescue (Chaipatikul et al., 2003;Noorwez et al., 2003;Wü ller et al., 2004;Fan et al., 2005;Robert et al., 2005b;Hawtin, 2006). It is a consistent requirement that ligands can penetrate into the cell; the pharmacochaperone effect is concentration-dependent in a range commensurate with the affinity value of the surface-exposed receptor. Retention in the endoplasmic reticulum (ER) or a later compartment along the secretory pathway means that the receptor has not acquired the proper native conformation. Pharmacochaper...

The adenosine A 2A receptor and the dopamine D 2 receptor are prototypically coupled to G s and G i /G o , respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor-G protein coupling. We tested this model in PC12 cells which endogenously express A 2A receptors. The human D 2 receptor was introduced into PC12 cells by stable transfection. A 2A -agonistmediated inhibition of D 2 agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D 2 agonist quinpirole depended on the expression level of the D 2 receptor: at low and high receptor levels, the A 2A -agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A 2A -receptor-mediated cAMP formation was inhibited by other G i /G o -coupled receptors that were either endogenously present (P 2y12 -like receptor for ADP) or stably expressed after transfection (A 1 adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca 2+ channels were inhibited by the endogenous P 2Y receptor and by the heterologously expressed A 1 receptor but not by the D 2 receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D 2 and A 2A receptors are closely associated, but they highlight the fact that this interaction can also support synergism.

The amphipathic peptide duramycin is in clinical development for the treatment of cystic fibrosis. It is deposited in cellular membranes where it binds to phosphatidylethanolamine. Duramycin may thereby change the biophysical membrane properties and perturb the function of ion channels. If so, in heart tissue, its application carries the risk to elicit cardiac arrhythmias. In fact, premature ventricular complexes were observed in the electrocardiogram during toxicological testing in dogs. To study the arrhythmogenic potential of duramycin, we investigated its effects on currents through voltage-gated hERG potassium, sodium, and calcium channels in native cells, and using a heterologous expression system, by means of the whole-cell patch clamp technique; duramycin bath concentrations between 1 nM and 0.1 microM did not generate any effects on these currents. Concentrations >or=0.3 microM, however, reduced the amplitudes of all investigated currents. Moreover, sodium current fast inactivation kinetics was slowed in the presence of duramycin. A further rise in duramycin bath concentration (>or=3.3 microM) induced a leak current consistent with pore formation. The reported effects of duramycin on ion channel function are likely to arise from a change in the biophysical properties of the membrane rather than from a specific interaction of the peptide with ion channel proteins. Under therapeutic conditions (i.e., administration via inhalation), duramycin plasma concentrations are below 0.5 nM. Thus, upon inhalation, duramycin has a large safety margin and is highly unlikely to elicit arrhythmias.