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The C terminus of the ␤ 2 -adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the distance between two positions on the ␤ 2 -AR C terminus and cysteine 265 (Cys-265) at the cytoplasmic end of transmembrane domain 6. The donor fluorophore FlAsH (Fluorescein Arsenical Helix binder) was attached to a CCPGCC motif introduced at position 351-356 in the proximal C terminus or at the distal C terminus. An acceptor fluorophore, Alexa Fluor 568, was attached to Cys-265. FRET analyses revealed that the average distances between Cys-265 and the proximal and distal FlAsH sites were 57 and 62 Å , respectively. These relatively large distances suggest that the C terminus is in an extended, relatively unstructured conformation. Nevertheless, we observed ligand-specific changes in FRET. All ligands induced an increase in FRET between the proximal C-terminal FlAsH site and Cys-265. Ligands that have been shown to induce arrestin-dependent ERK activation, including the catecholamine agonists and the inverse agonist ICI118551, led to a decrease in FRET between the distal FlAsH site and Cys-265, whereas other ligands had no effect or induced a small increase in FRET. Taken together the results provide new insight into the structure of the C terminus of the ␤ 2 -AR as well as ligand-induced conformational changes that may be relevant to arrestin-dependent regulation and signaling.G protein-coupled receptors (GPCRs) 3 are versatile membrane proteins that regulate a wide variety of physiological functions. They respond to a large array of structurally diverse ligands and are the largest group of targets for drug discovery. Structure/function analysis has identified amino acids important for G protein coupling and ligand binding for several well characterized GPCRs, including the ␤-2 adrenoceptor (␤ 2 -AR) (1). Moreover, many of these studies provide support for the accuracy of three-dimensional models of GPCRs based on the high resolution structure of bovine rhodopsin (2). However, few studies have directly addressed the mechanism by which diffusible ligands activate G proteincoupled receptors.The most detailed information about structural changes associated with activation of a GPCR come from studies of rhodopsin. This is in part owing to its natural abundance and biochemical stability relative to other GPCRs. Electron paramagnetic resonance spectroscopy studies provide evidence that photoactivation of rhodopsin involves a rotation and tilting of transmembrane segment 6 (TM6) relative to TM3 (3). Lightinduced conformational changes have also been observed in the cytoplasmic domain spanning TM1 and TM2, and the cytoplasmic end of TM7 (4 -6).Although rhodopsin has long been used as a model system for GPCR activation, it is unique among GPCRs because of...

Anti-GD2 mAbs, acting via antibody-dependent cell-mediated cytotoxicity, may enhance the effects of chemotherapy. This pilot trial investigated a fixed dose of a unique anti-GD2 mAb, hu14.18K322A, combined with chemotherapy, cytokines, and haploidentical natural killer (NK) cells. Children with recurrent/refractory neuroblastoma received up to six courses of hu14.18K322A (40 mg/m/dose, days 2-5), GM-CSF, and IL2 with chemotherapy: cyclophosphamide/topotecan (courses 1,2), irinotecan/temozolomide (courses 3,4), and ifosfamide/carboplatin/etoposide (courses 5,6). Parentally derived NK cells were administered with courses 2, 4, and 6. Serum for pharmacokinetic studies of hu14.18K322A, soluble IL2 receptor alpha (sIL2Rα) levels, and human antihuman antibodies (HAHA) were obtained. Thirteen heavily pretreated patients (9 with prior anti-GD2 therapy) completed 65 courses. One patient developed an unacceptable toxicity (grade 4 thrombocytopenia >35 days). Four patients discontinued treatment for adverse events (hu14.18K322A allergic reaction, viral infection, surgical death, second malignancy). Common toxicities included grade 3/4 myelosuppression (13/13 patients) and grade 1/2 pain (13/13 patients). Eleven patients received 29 NK-cell infusions. The response rate was 61.5% (4 complete responses, 1 very good partial response, 3 partial responses) and five had stable disease. The median time to progression was 274 days (range, 239-568 days); 10 of 13 patients (77%) survived 1 year. Hu14.18K322A pharmacokinetics was not affected by chemotherapy or HAHA. All patients had increased sIL2Rα levels, indicating immune activation. Chemotherapy plus hu14.18K322A, cytokines, and NK cells is feasible and resulted in clinically meaningful responses in patients with refractory/recurrent neuroblastoma. Further studies of this approach are warranted in patients with relapsed and newly diagnosed neuroblastoma. .