Citations

The secretin receptor is a prototypic class B G protein-coupled receptor that is activated by binding of its natural peptide ligand. The signaling effects of this receptor are mediated by coupling with Gs, which activates cAMP production, and Gq, which activates intracellular calcium mobilization. We have explored the molecular basis for the coupling of each of these G proteins to this receptor using systematic site-directed mutagenesis of key residues within each of the intracellular loop regions, and studying ligand binding and secretin-stimulated cAMP and calcium responses. Mutation of a conserved histidine in the first intracellular loop (H157A and H157R) markedly reduced cell surface expression, resulting in marked reduction in cAMP and elimination of measurable calcium responses. Mutation of an arginine (R153A) in the first intracellular loop reduced calcium, but not cAMP responses. Mutation of a dibasic motif in the second intracellular loop (R231A/K232A) had no significant effects on any measured responses. Mutations in the third intracellular loop involving adjacent lysine and leucine residues (K302A/L303A) or two arginine residues separated by a leucine and an alanine (R318A/R321A) significantly reduced cAMP responses, while the latter also reduced calcium responses. Additive effects were elicited by combining the effective mutations, while combining all the effective mutations resulted in a construct that continued to bind secretin normally, but that elicited no significant cAMP or calcium responses. These data suggest that, while some receptor determinants are clearly shared, there are also distinct determinants for coupling with each of these G proteins.

The role of radiotherapy for local control of marginally resected, unresectable, and recurrent giant cell tumors of bone (GCToB) has not been well defined. The number of patients affected by this rare disease is low. We present a series of 58 patients with biopsy proven GCToB who were treated with radiation therapy. A retrospective review of the role of radiotherapy in the treatment of GCToB was conducted in participating institutions of the Rare Cancer Network. Eligibility criteria consisted of the use of radiotherapy for marginally resected, unresectable, and recurrent GCToB. Fifty-eight patients with biopsy proven GCToB were analyzed from 9 participating North American and European institutions. Forty-five patients had a primary tumor and 13 patients had a recurrent tumor. Median radiation dose was 50 Gy in a median of 25 fractions. Indication for radiation therapy was marginal resection in 33 patients, unresectable tumor in 13 patients, recurrence in 9 patients and palliation in 2 patients. Median tumor size was 7.0 cm. A significant proportion of the tumors involved critical structures. Median follow-up was 8.0 years. Five year local control was 85%. Of the 7 local failures, 3 were treated successfully with salvage surgery. All patients who received palliation achieved symptom relief. Five year overall survival was 94%. None of the patients experienced grade 3 or higher acute toxicity. This study reports a large published experience in the treatment of GCToB with radiotherapy. Radiotherapy can provide excellent local control for incompletely resected, unresectable or recurrent GCToB with acceptable morbidity.

Background— Pulmonary arterial hypertension (PAH) is a rare but important cause of morbidity and mortality in children. Methods and Results— We analyzed data from 216 patients ≤18 years of age at diagnosis who were enrolled in the Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL). Median age at diagnosis and enrollment was 7 and 15 years, respectively. The most frequent presenting symptom was dyspnea (idiopathic/familial PAH, 53%; PAH associated with congenital heart disease, 30%). Presyncope/syncope was more frequent in patients with idiopathic PAH/familial PAH (36%) than in those with PAH associated with congenital heart disease (4%). At diagnosis, mean pulmonary artery pressure and pulmonary vascular resistance index were 56 mm Hg and 17 Wood units · m 2 , respectively. Five-year survival from diagnosis for the overall cohort was 74±6%, with no significant difference between the idiopathic PAH/familial PAH (n=122, 75±7%) and PAH associated with congenital heart disease (n=77, 71±13%) cohorts ( P =0.53). Older age at diagnosis was the only variable significantly associated with decreased survival from diagnosis. Variables at enrollment that were significantly associated with decreased survival from enrollment included higher pulmonary vascular resistance index, lower-weight z scores, and familial PAH. Additional variables at enrollment, identified in a secondary analysis, that were marginally associated with increased survival from enrollment included acute vasoreactivity (adaptation of conventional pediatric definition; P =0.087) and lower brain natriuretic peptide ( P =0.060). None of the 22 patients who were acute responders treated with high-dose calcium channel blockade as monotherapy or combination therapy died within 5 years of diagnosis. Conclusion— Using REVEAL, we identified key predictors of survival in childhood PAH. Refining these prognostic parameters should help clinicians improve outcomes. Clinical Trial Registration— URL: www.clinicaltrials.gov . Unique identifier: NCT00370214.

Receptor ligands, identified as antagonists, based on the absence of stimulation of signaling, can rarely stimulate receptor internalization. d-Tyr-Gly-[(Nle28,31,d-Trp30)CCK-26–32]-2-phenylethyl ester (d-Trp-OPE) is such a ligand that binds to the cholecystokinin (CCK) receptor and stimulates internalization. Here, the molecular basis of this trafficking event is explored, with the assumption that ligand binding initiates conformational change, exposing an epitope to direct endocytosis. Ligand-stimulated internalization was studied morphologically using fluorescent CCK and d-Trp-OPE. d-Trp-OPE occupation of Chinese hamster ovary cell receptors stimulated internalization into the same region as CCK. Arrestin-biased action was ruled out using morphological translocation of fluorescent arrestin 2 and arrestin 3, moving to the membrane in response to CCK, but not d-Trp-OPE. Possible roles of the carboxyl terminus were studied using truncated receptor constructs, eliminating the proline-rich distal tail, the serine/threonine-rich midregion, and the remainder to the vicinal cysteines. None of these constructs disrupted d-Trp-OPE-stimulated internalization. Possible contributions of transmembrane segments were studied using competitive inhibition with peptides that also had no effect. Intracellular regions were studied with a similar strategy using coexpressing cell lines. Peptides corresponding to ends of each loop region were studied, with only the peptide at the carboxyl end of the third loop inhibiting d-Trp-OPE-stimulated internalization but having no effect on CCK-stimulated internalization. The region contributing to this effect was refined to peptide 309–323, located below the recognized G protein-association motif. While a receptor in which this segment was deleted did internalize in response to d-Trp-OPE, it exhibited abnormal ligand binding and did not signal in response to CCK, suggesting an abnormal conformation and possible mechanism of internalization distinct from that being studied. This interpretation was further supported by the inability of peptide 309–323 to inhibit its d-Trp-OPE-stimulated internalization. Thus the 309–323 region of the type 1 CCK receptor affects antagonist-stimulated internalization of this receptor, although its mechanism and interacting partner are not yet clear.