- Email: [email protected]
Characterization and molecular mechanism of GIP receptor internalization
Abstracts / Regulatory Peptides 164 (2010) 35–51
Structural analysis of the six most potent purified insulinotropic fractions (39, 43, 49, 52, 53 and 55) revealed peptides with molecular masses of 1537.0–2417.3 Da, with amino acid sequences of GIKEFAHSLGKFGKAFVGGILNQ, GMASKAGSVLGKVAKVALKAAL.NH 2 , GLGSVLGKALKIGANLL.NH 2 , GIGSALAKAAKLVAGIV.NH 2 , GWASKIAQTLGKMAKVGLQELIQPK and GLGSLVGNALRIGAKLL.NH2. These peptides are related to magainin, peptide-glycine-leucine-amide (PGLa), caerulin-precursor-fragment (CPF) and xenopsin-precursorfragment (XPF) previously isolated from Xenopus laevis. Discussion: The skin secretions of Xenopus amieti frog contain multiple non-toxic insulin-releasing peptides which could be further investigated and developed into novel anti-diabetic drugs.
doi:10.1016/j.regpep.2010.07.090
A007 Selective processing of chromogranin A in rectal carcinoid tumours: An immunohistochemical study with region-specific antibodies G.M. Portela-Gomesa, L. Grimeliusb, M. Stridsbergc a Department of Gastroenterology, Lisbon University, Portugal b Departments of Genetics and Pathology, Uppsala University, Uppsala, Sweden c Department of Medical Sciences, Uppsala University, Uppsala, Sweden Hindgut carcinoid tumours of the L-cell type are usually nonreactive or display weak or focal immunoreactivity to chromogranin (Cg) A. The antibodies most commonly used in routine histopathology are raised either to epitopes localized in the sequence 250–284 (N-terminal pancreastatin; monoclonal antibody LK2H10) or to the C-terminal half of the CgA molecule. In the present study 12 rectal carcinoid tumours, 10 of the L-cell type and 2 of the EC cell type, were analyzed immunohistochemically regarding the expression of antibodies raised against 13 different epitopes of the CgA molecule. The results revealed a varying expression of region-specific CgA epitopes regarding both the frequency of immunoreactive cells and intensity of immunoreactivity. The LK2H10 antibody immunostained weakly and/or focally only few cells in 4 of the 10 L-cell type tumours and strongly the two EC-cell tumours. Most neoplastic cells were strongly stained by CgA 17–38 (vasostatin) in all tumours except one Lcell tumour; CgA 1–17 (N-terminal vasostatin) was expressed in 6 of the L-cell type tumours. CgA 411–424 (C-teminal parastatin) stained weakly all tumours except one L-cell tumour. On the other hand, CgA 361–372 (catestatin) was lacking in all L-cell type tumours, while CgA 238–247 and 375–384 appeared only in two of the L-cell tumours, however in a minority of cells. The expression of the other midportion epitopes of CgA varied, and was mostly scarce and focal in L-cell type rectal carcinoids, with few exceptions. The two EC-cell type tumours expressed all 13 CgA epitopes in a majority of neoplastic cells. In conclusion, L-cell type rectal carcinoid tumours expressed mainly and strongly N-terminal epitopes of the CgA molecule (vasostatins) but weakly the C-terminal (parastatin). This study shows that N-terminal CgA antibodies seem more useful for histopathological diagnosis of rectal carcinoids than the antibodies raised against other CgA regions. doi:10.1016/j.regpep.2010.07.091
A008 Characterization and molecular mechanism of GIP receptor internalization M. Laval1, R. Magnan, B. Masri, D. Fourmy1
37
Unité INSERM U-858, I2MR, équipe 13, CHU Rangueil, Bât. L3, BP 84225, 1 avenue Jean Poulhès, 31432 Toulouse Cedex 4, France 1 Tel.: + 33 561 32 30 57; fax: + 33 5 61 32 24 03. E-mail addresses: [email protected] (M. Laval), [email protected] (M.D. Fourmy). Introduction: Glucose-dependent insulinotropic polypeptide (GIP) is a polypeptide hormone which potentiates glucose-dependent insulin secretion in pancreatic β cells. However, the incretin function of GIP is severely down-regulated in patients with type 2 diabetes. So far, only regulation of rodent GIP receptor, the sequence of which differs from the human one, has been explored. Aims: To study internalization of the human GIP receptor and molecular mechanisms involved in order to have a better understanding of GIP receptor down-regulation and thus possibly explain failure of insulinotropic action of GIP during chronic hyperglycemia in humans. Methods: Internalization of GIP bound to its receptor expressed in HEK cells has been followed by confocal microscopy, and we characterized endocytosis pathway by identifying the role of clathrin-coated vesicles and dynamin. Furthermore the importance of beta-arrestins was investigated by BRET and confocal microscopy. HEK cells expressing human CCK2 receptors were used as a reference cell model for internalization. Results: Endocytosis of GIP bound to its receptor is a fast phenomenon occurring in the first 5 min. After prolonged stimulation, the ligand is observed in endosomes. GIPR is internalized through clathrin-coated pits and involved dynamin GTPase. Nevertheless, unlike CCK2R and most of GPCRs, human GIP receptor internalization is not dependent on beta-arrestin1 and 2 recruitment. Discussion: Although data were reported suggesting that phosphorylation of GIP receptor on its C-terminal region and betaarrestin1 recruitment may be important for receptor down-regulation, our data support that human GIP receptor internalization occurs though a mechanism independent of beta-arrestins. This mechanism is being investigated. doi:10.1016/j.regpep.2010.07.092
A009 Intestinal serotonin (5-HT) signalling is hormonally regulated via glucagon-like peptide 1 (GLP1) and PKA/MAPK activation M. Kidda, A. Kazberouka, F. Giovinazzaa, A. Timberlakea, R. Pfragnerb, I.M. Modlina a Gastrointestinal Pathobiology Research Group, Yale University School of Medicine, USA b Institute of Molecular Medicine, Medical University of Graz, Austria Background: Serotonin (5-HT)-secreting enterochromaffin (EC) cells and GLP-1 secreting L cells are chemosensory enteroendocrine cells that colocalize in the GI tract and play key roles in regulating the response of the GI tract to the contents of the lumen (i.e. motility and islet secretion). Aims: Evaluate whether an interactive functional relationship existed between L and EC cells. Methods: NCI-H716 (L cells) and KRJ-1 (EC cells) as well as duodenal mucosa and isolated normal EC cells were used to study hormone release. Receptor expression was identified using real-time PCR and western blot. 5-HT and GLP1 secretion was assessed (ELISA) and signaling pathways investigated (cAMP ELISA and MAPK phosphorylation). Results: EC cells express GLP receptor mRNA and protein and L cells express 5-HT1A and 5-HT2A receptors. The long-acting GLP1 agonist, exendin-4, decreased 5-HT secretion from normal and neoplastic EC cells as well as from whole mucosa (25%–40% decrease,
Structural analysis of the six most potent purified insulinotropic fractions (39, 43, 49, 52, 53 and 55) revealed peptides with molecular masses of 1537.0–2417.3 Da, with amino acid sequences of GIKEFAHSLGKFGKAFVGGILNQ, GMASKAGSVLGKVAKVALKAAL.NH 2 , GLGSVLGKALKIGANLL.NH 2 , GIGSALAKAAKLVAGIV.NH 2 , GWASKIAQTLGKMAKVGLQELIQPK and GLGSLVGNALRIGAKLL.NH2. These peptides are related to magainin, peptide-glycine-leucine-amide (PGLa), caerulin-precursor-fragment (CPF) and xenopsin-precursorfragment (XPF) previously isolated from Xenopus laevis. Discussion: The skin secretions of Xenopus amieti frog contain multiple non-toxic insulin-releasing peptides which could be further investigated and developed into novel anti-diabetic drugs.
doi:10.1016/j.regpep.2010.07.090
A007 Selective processing of chromogranin A in rectal carcinoid tumours: An immunohistochemical study with region-specific antibodies G.M. Portela-Gomesa, L. Grimeliusb, M. Stridsbergc a Department of Gastroenterology, Lisbon University, Portugal b Departments of Genetics and Pathology, Uppsala University, Uppsala, Sweden c Department of Medical Sciences, Uppsala University, Uppsala, Sweden Hindgut carcinoid tumours of the L-cell type are usually nonreactive or display weak or focal immunoreactivity to chromogranin (Cg) A. The antibodies most commonly used in routine histopathology are raised either to epitopes localized in the sequence 250–284 (N-terminal pancreastatin; monoclonal antibody LK2H10) or to the C-terminal half of the CgA molecule. In the present study 12 rectal carcinoid tumours, 10 of the L-cell type and 2 of the EC cell type, were analyzed immunohistochemically regarding the expression of antibodies raised against 13 different epitopes of the CgA molecule. The results revealed a varying expression of region-specific CgA epitopes regarding both the frequency of immunoreactive cells and intensity of immunoreactivity. The LK2H10 antibody immunostained weakly and/or focally only few cells in 4 of the 10 L-cell type tumours and strongly the two EC-cell tumours. Most neoplastic cells were strongly stained by CgA 17–38 (vasostatin) in all tumours except one Lcell tumour; CgA 1–17 (N-terminal vasostatin) was expressed in 6 of the L-cell type tumours. CgA 411–424 (C-teminal parastatin) stained weakly all tumours except one L-cell tumour. On the other hand, CgA 361–372 (catestatin) was lacking in all L-cell type tumours, while CgA 238–247 and 375–384 appeared only in two of the L-cell tumours, however in a minority of cells. The expression of the other midportion epitopes of CgA varied, and was mostly scarce and focal in L-cell type rectal carcinoids, with few exceptions. The two EC-cell type tumours expressed all 13 CgA epitopes in a majority of neoplastic cells. In conclusion, L-cell type rectal carcinoid tumours expressed mainly and strongly N-terminal epitopes of the CgA molecule (vasostatins) but weakly the C-terminal (parastatin). This study shows that N-terminal CgA antibodies seem more useful for histopathological diagnosis of rectal carcinoids than the antibodies raised against other CgA regions. doi:10.1016/j.regpep.2010.07.091
A008 Characterization and molecular mechanism of GIP receptor internalization M. Laval1, R. Magnan, B. Masri, D. Fourmy1
37
Unité INSERM U-858, I2MR, équipe 13, CHU Rangueil, Bât. L3, BP 84225, 1 avenue Jean Poulhès, 31432 Toulouse Cedex 4, France 1 Tel.: + 33 561 32 30 57; fax: + 33 5 61 32 24 03. E-mail addresses: [email protected] (M. Laval), [email protected] (M.D. Fourmy). Introduction: Glucose-dependent insulinotropic polypeptide (GIP) is a polypeptide hormone which potentiates glucose-dependent insulin secretion in pancreatic β cells. However, the incretin function of GIP is severely down-regulated in patients with type 2 diabetes. So far, only regulation of rodent GIP receptor, the sequence of which differs from the human one, has been explored. Aims: To study internalization of the human GIP receptor and molecular mechanisms involved in order to have a better understanding of GIP receptor down-regulation and thus possibly explain failure of insulinotropic action of GIP during chronic hyperglycemia in humans. Methods: Internalization of GIP bound to its receptor expressed in HEK cells has been followed by confocal microscopy, and we characterized endocytosis pathway by identifying the role of clathrin-coated vesicles and dynamin. Furthermore the importance of beta-arrestins was investigated by BRET and confocal microscopy. HEK cells expressing human CCK2 receptors were used as a reference cell model for internalization. Results: Endocytosis of GIP bound to its receptor is a fast phenomenon occurring in the first 5 min. After prolonged stimulation, the ligand is observed in endosomes. GIPR is internalized through clathrin-coated pits and involved dynamin GTPase. Nevertheless, unlike CCK2R and most of GPCRs, human GIP receptor internalization is not dependent on beta-arrestin1 and 2 recruitment. Discussion: Although data were reported suggesting that phosphorylation of GIP receptor on its C-terminal region and betaarrestin1 recruitment may be important for receptor down-regulation, our data support that human GIP receptor internalization occurs though a mechanism independent of beta-arrestins. This mechanism is being investigated. doi:10.1016/j.regpep.2010.07.092
A009 Intestinal serotonin (5-HT) signalling is hormonally regulated via glucagon-like peptide 1 (GLP1) and PKA/MAPK activation M. Kidda, A. Kazberouka, F. Giovinazzaa, A. Timberlakea, R. Pfragnerb, I.M. Modlina a Gastrointestinal Pathobiology Research Group, Yale University School of Medicine, USA b Institute of Molecular Medicine, Medical University of Graz, Austria Background: Serotonin (5-HT)-secreting enterochromaffin (EC) cells and GLP-1 secreting L cells are chemosensory enteroendocrine cells that colocalize in the GI tract and play key roles in regulating the response of the GI tract to the contents of the lumen (i.e. motility and islet secretion). Aims: Evaluate whether an interactive functional relationship existed between L and EC cells. Methods: NCI-H716 (L cells) and KRJ-1 (EC cells) as well as duodenal mucosa and isolated normal EC cells were used to study hormone release. Receptor expression was identified using real-time PCR and western blot. 5-HT and GLP1 secretion was assessed (ELISA) and signaling pathways investigated (cAMP ELISA and MAPK phosphorylation). Results: EC cells express GLP receptor mRNA and protein and L cells express 5-HT1A and 5-HT2A receptors. The long-acting GLP1 agonist, exendin-4, decreased 5-HT secretion from normal and neoplastic EC cells as well as from whole mucosa (25%–40% decrease,