title: A natural inactivating mutant of human glucagon receptor exhibits multiple abnormalities in processing and signaling.
abstract: OBJECTIVE
  To elucidate the pathogenetic mechanisms of a mutant P86S glucagon receptor (GCGR) in causing a novel human disease (Mahvash disease).
  
  
  METHODS
  Enhanced green fluorescent protein (EGFP)-tagged WT and P86S GCGR were expressed in HEK 293 or H1299 cells either transiently or stably. Receptor localization and internalization, and cell apoptosis were studied by fluorescence microscopy, and calcium signaling by Rhod-3 labeling. Gene expression was assayed by RT-PCR or Western blot. Cell fate was determined by live cell imaging.
  
  
  RESULTS
  Unlike WT GCGR, P86S was partially localized to the plasma membrane and partially in the cytoplasm as previously reported and did not undergo internalization upon glucagon treatment. P86S did not elicit calcium response after treatment with 1 μM glucagon. Cells transiently expressing P86S exhibited more apoptosis than those expressing WT GCGR (18.3% vs 2.1%, P<0.05) but the X-box binding protein 1 mRNA cleavage, a marker of endoplasmic reticulum (ER) stress, was not evident, suggesting that the apoptosis did not result from ER stress. Cells stably expressing P86S did not exhibit apoptosis and a quarter of them harbored a novel inclusion body-like circular structure that was marked by P86S and ER residential proteins. These circular ER bodies were not seen in cells expressing WT GCGR or transiently expressing P86S and were not affected by treatment with proteasome inhibitor or microtubule depolymerizer, suggesting that they do not represent aggresome structures. The circular ER bodies could fuse and split to form new bodies.
  
  
  CONCLUSIONS
  The naturally-occurring P86S mutant GCGR exhibits abnormal receptor internalization and calcium mobilization, and causes apoptosis. The novel dynamic circular ER bodies may be adaptive in nature to nullify the toxic effects on P86S. These findings provide further insights into the pathogenetic mechanisms of Mahvash disease.
authors.affiliation: Division of Endocrinology, Cedars-Sinai Medical Center, Los Angeles, California, USA. run.yu@cshs.org
authors.forename: Run
authors.initials: R
authors.lastname: Yu
authors.forename: Kolja
authors.initials: K
authors.lastname: Wawrowsky
authors.forename: Cuiqi
authors.initials: C
authors.lastname: Zhou
mesh_terms: D017209:Apoptosis / Q000235:genetics; D020013:Calcium Signaling / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D005934:Glucagon / Q000097:blood*; D050416:Glucagon-Secreting Cells / Q000473:pathology; D006801:Humans; D006965:Hyperplasia; D007003:Hypoglycemia / Q000235:genetics; D020125:Mutation, Missense; D009386:Neoplastic Syndromes, Hereditary / Q000235:genetics*; D018358:Neuroendocrine Tumors / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000235:genetics*; D017354:Point Mutation; D018027:Receptors, Glucagon / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology
chemical_list: D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins; D005934:Glucagon; D000242:Cyclic AMP