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Identification of a key residue involved in the activation of the cholecystokinin B receptor
Al144 AGAABSTRACTS
GASTROENTEROLOGYVol. 114, No. 4
G4681 IDENTIFICATION OF A KEY RESIDUE INVOLVED IN THE ACTIVATION OF THE CHOLECYSTOKININ B RECEPTOR. C. Gal~s, C. Escrieut, D. Fourmy, B. Maigret*, L. Moroder~, N. Vaysse and S.S. Pgir0t. INSERM U 151, CHU de Rangueil, 31403 Toulouse Cx-4, France. *CNRS UA 510, Nancy, France. §Max Planck Institut Martinsried, Germany. The cholecystokinin B receptor (CCKBR) is a member of the G-protein coupled receptor family (GPCR) that stimulates inositol phosphates (IP) production via coupling to a pertussis toxin-insensitive G protein presumably of the Gq family. In the course to determine the molecular processes in the CCKBR that lead to phospholipase C (PLC) activation, we mutated several residues in the CCKBR reported important in others GPCR for mediating IP production. The mutation of one of these residues localized in the transmembrane domains of CCKBR completely abolished CCK-9 stimulated IP production in Cos ceils expressing the mutant receptor compared to ceils expressing the wild type CCKBR. In contrast, this mutation did not affect significantly the high affinity binding and the binding capacity of I~I-BH-CCK-9 compared to the wild type CCKBR: Kd = 2.51 nM, Bmax = 0.61 pmol/106 cells and Kd = 0.96 nM and Bmax = 0.61 pmol/106 cells respectively, indicating normal expression at the cells surface. In contrast this mutation increased the affinity of the non-peptide CCKBR antagonists L365 260 and PD135 158 of 3 and 8-fold respectively, suggesting that this residue may participate to the antagonist binding site. lzSI-BH-CCK-9 binding in presence of increasing concentrations of the non-hydrolyzable analog of GTP (GTP .S) on Cos cells membranes expressing the wild type and the mutated receptor reveales that the affinity of CCK-9 is not affected by GTPrS in the mutated receptor while in the wild type CCKBR the affinity of CCK-9 is strongly reduced. These results suggest that in the mutated receptor the coupling to the G protein that mediates PLC activation is strongly affected and may explain the lack of 1P production. These data should help to progress in the study of the molecular mechanisms involved in signal transduction that still remain poorly understood in the CCKBR. r
.
.
• G4682 CONSTITUTIVE ACTIVATION OF THE HUMAN VIP1/PACAP RECEPTOR, A MEMBER OF THE NEW CLASS II FAMILY OF G PROTEIN-COUPLED RECEPTORS. P. Gaudin, J.J. Maoret, A. Couvineau, C. Rouyer-Fessard, M. Laburthe, Paris, France.
The human VIPI/PACAP receptor belongs to the new class II subfamily of G protein-coupled receptors. We report here that the specific change by sitedirected mutagenesis of a strictly conserved histidine (H) into arginine (R) at position 178 of the human VIP1 receptor (junction between the first intracellular loop and second membrane-spanning domain) resulted in its constitutive, ligand-independent, activation with respect to cAMP production. Transfection of the H178R mutant into Cos cells resulted in a 3.5 foldincrease in the cAMP level as compared to cells transfected with the wild type receptor or the pcDNA3 vector alone. This increase was proportional to the amount of transfected eDNA encoding the H178R mutant. The H178R mutant exhibited an otherwise normal cAMP response to VIP in terms of potency and efficacy as well as a dissociation constant similar to that of the wild type receptor as determined by binding studies. Other mutants at position 178 such as H178K, H178A and H178D were not constitutively activated. They were otherwise expressed at the cell surface of transfected nonpermeabilized Cos ceils as assessed by immunofluorescence studies and antibody binding experiments carried out with receptors that were epitope-tagged in the N-terminal extracellular domain. Double mutants were then constructed in which the H178R mutation was associated with a point mutation in the the N-terminal extracellular domain that totally abolished VIP binding or VIP-stimulated cAMP production i.e. E36A or D68A. The corresponding double mutants H178R-E36A and HI78R-D68A were no longer constitutively activated. A control double mutant (HI78R-D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP, was still constitutively activated. Our findings demonstrate that constitutive activation of the VIPI/PACAP receptor by mutation of H178 into R requires the functional integrity of the N-terminal extracellular VIP binding domain. They might provide interesting generalities about the activation process in the class II family of G protein-coupled receptors. • G4683 A PROSPECTIVE STUDY OF GASTRIC LOCALIZATION IN PATIENTS WITH ZOLLINGER-ELLISON SYNDROME (ZES) WITH SOMATOSTATIN RECEPTOR SCINTIGRAPHY (SRS) AND ITS ASSOCIATION WITH THE PRESENCE OF GASTRIC CARCINOID TUMORS. F. Gibril, J.C. Reynolds, C.C. Chen, J. Serrano, S.U. Goebel, F. Yu, I.A. Lubensky, NIH, Bethesda, MD; J.A. Norton, UCSF Medical Center, San Francisco, CA; R.T. Jensen, NIH, Bethesda, MD.
Hypergastrinemic states cause an increase in the occurrence of gastric carcinoid tumors which can be difficult to identify. Recent studies show most carcinoid tumors and pancreatic endocrine tumors (PETs) have a high density of somatostatin receptors and that SRS is a very sensitive method to localize them. The purpose of the present study was to determine how frequently SRS localized to the stomach in patients with ZES and whether there was an association with the presence of gastric carcinoid tumors. 150 consecutive patients with ZES were studied prospectively with 32 having MEN-I, and 118 patients not having MEN-I. All patients underwent SRS with 6 mCi of [mIn-DTPA-DPhel]octreotide with SPECT imaging and upper
gastrointestinal endoscopy with Jumbo biopsies of any gastric abnormalities or if no abnormalities were seen two random biopsies of the gastric body were taken. Gastric carcinoid tumors were identified in 131150 (9%) of patients, 11/32 (34%) patients with MEN-I and 2/118 (2%) of patients without MEN-1. SRS demonstrated gastric localization in 121150 (8%) of patients, 10132 (31%) with MEN-I and 21118 (2%) of patients without MEN-I. In all 12 patients with positive gastric localization the localization was diffuse over the body of the stomach and in addition 4 of these patients had focal intense areas of localization. The focal intense localization was only seen in 4 patients with MEN-I. In the 32 patients with ZES and MEN-I, 9 of the I 1 (82%) patients with gastric carcinoid tumors had a positive SRS in the stomach. Of the 118 patients without MEN-I neither of the 2 patients with a gastric carcinoid had gastric uptake on the SRS and 2 of the 116 (2%) patients without MEN-I or a gastric carcinoid had uptake in the stomach. These results demonstrate that with SRS gastric localization is seen in 8% of ZES patients and occurs much more frequently (p <0.0001) in patients with MEN-I with ZES (31% vs 2%). Furthermore, gastric SRS localization was significantly (p<0.0001) associated with the presence of gastric carcinoid tumors (69% vs 2%). These results suggest SRS may be a useful method to localize gastric carcinoid tumors and conversely, it is important to remember in patients with ZES and/or MEN-I that SRS localization to the upper abdomen may represent a gastric carcinoid, not a primary or metastatic gastrinoma. G4684 AN AGGRESSIVE AND NONAGGRESSIVE FORM OF DISEASE EXISTS IN PATIENTS WITH MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 AND ZOLLINGER-ELLISON SYNDROME (ZES). F. Gibril, J. Serrano, F. Yu, S.U. Goebel, R.T. Jensen, NIH, Bethesda, MD.
Gastrinomas occur as a sporadic form (62-80%) and as a familial form with Multiple Endocrine Neoplasia-type 1 (MEN-I). A recent study shows that gastrinomas, in patients primarily with sporadic disease, can pursue an aggressive (25%) or nonaggressive form. In Older studies patients with MEN-I with ZES had a better prognosis and the tumor was thought to have an indolent course. However, a recent case report proposes aggressive forms of tumor growth may occur also in patients with MEN-I with ZES. The purpose of the present study was to determine whether an aggressive form of the disease occurs in patients with MEN-I, the percentage of MEN-I so affected, and whether these patients can be distinguished by laboratory or clinical parameters. All patients with MEN-I and ZES followed at the NIH were included. Patients underwent conventional imaging studies yearly (CT scan, ultrasound, MRI) and angiography if the conventional imaging results were unclear. In the last 3 years, yearly SRS was performed on all patients. If liver metastases were present patients had imaging studies every 3-6 months. Patients were divided into 2 groups, those with aggressive disease [rapid tumor growth (>25%/mo) which continued over subsequent admissions], or non aggressive disease. Patients had been followed from 4 months to 25 years. Five of 42 patients (12%) had aggressive tumor growth and 37142 (88%) did not have aggressive growth with a follow-up time of 8.4 and 7.6 years, respectively. Patients with and without aggressive disease did not differ in age, gender, duration of disease, basal acid output, percentage that underwent surgery or tumor location. Patients with aggressive disease more frequently had liver metastases (4/5=80% vs 3/37=8%, p=0.002), bone metastases (2/5=40% vs 0/37=0%, p=0.013) and had higher fasting gastrin levels (p <0.01). The difference in the rate of death from the gastrinoma [1/5 (20%) vs 0/31 (0%)] was not significant (p=0.11). These results demonstrate that in 12% of patients with MEN-I the gastrinoma pursues an aggressive course. These patients are significantly more likely to have liver, bone metastases and to have higher gastrin levels. At present patients with the aggressive form can not be distinguished from those without it unequivocally by any clinical or laboratory characteristic. Therefore, it will be important to compare these two groups in future studies for any differences in tumor markers or molecular abnormalities that will allow earlier anti-tumor strategies to be instituted in patients with aggressive disease. Furthermore, because a proportion of patients with ZES with MEN-I also have aggressive disease, these patients need to be followed carefully to attempt to detect this as soon as possible to institute antitumor treatment. G4685 POLYAMINES MODULATE PHAS-I GENE EXPRESSION IN AN INTESTINAL CELL LINE. John Gill, Karin Suess, ER Seidel. Department of Physiology, School of Medicine, East Carolina University, Greenville, NC. Ornithine decarboxylase (ODC), one of the rate limiting enzymes in polyamine biosynthesis, is a highly inducible enzyme whose activity is strictly regulated. Over-expression of ODC leads to neoplastic transformation. Regulation appears to posttranscriptional; ODC protein activity may increase 25-fold after growth factor stimulation with no change in steady state ODC mRNA levels. PHAS-I is a heat stable protein which may be a regulator of protein synthesis in response to agents which activate MAP kinases. PHAS-I or elF-4E binding protein limits the availability of elF-4E, a key component of the elF-4F translation initiation complex. Treatment of IEC-6 cells with PD 098059, a MEK 1 inhibitor, blocked the induction of ODC in repsonse to FBS by 90% confirming the potential involvement of MAP kinases in regulation of ODC translation. Cells were polyamine-depleted by 72 hr treatment with difluoromethylornithine, an inhibitor of ODC, followed by readdition of 10 uM putrescine. Total RNA was collected over the next 24 hr and ODC transcript level determined by ribonuclease protection assay. PHAS-I transcript levels began to fall 4 hr after putrescine and reached a nadir at 12 hr
GASTROENTEROLOGYVol. 114, No. 4
G4681 IDENTIFICATION OF A KEY RESIDUE INVOLVED IN THE ACTIVATION OF THE CHOLECYSTOKININ B RECEPTOR. C. Gal~s, C. Escrieut, D. Fourmy, B. Maigret*, L. Moroder~, N. Vaysse and S.S. Pgir0t. INSERM U 151, CHU de Rangueil, 31403 Toulouse Cx-4, France. *CNRS UA 510, Nancy, France. §Max Planck Institut Martinsried, Germany. The cholecystokinin B receptor (CCKBR) is a member of the G-protein coupled receptor family (GPCR) that stimulates inositol phosphates (IP) production via coupling to a pertussis toxin-insensitive G protein presumably of the Gq family. In the course to determine the molecular processes in the CCKBR that lead to phospholipase C (PLC) activation, we mutated several residues in the CCKBR reported important in others GPCR for mediating IP production. The mutation of one of these residues localized in the transmembrane domains of CCKBR completely abolished CCK-9 stimulated IP production in Cos ceils expressing the mutant receptor compared to ceils expressing the wild type CCKBR. In contrast, this mutation did not affect significantly the high affinity binding and the binding capacity of I~I-BH-CCK-9 compared to the wild type CCKBR: Kd = 2.51 nM, Bmax = 0.61 pmol/106 cells and Kd = 0.96 nM and Bmax = 0.61 pmol/106 cells respectively, indicating normal expression at the cells surface. In contrast this mutation increased the affinity of the non-peptide CCKBR antagonists L365 260 and PD135 158 of 3 and 8-fold respectively, suggesting that this residue may participate to the antagonist binding site. lzSI-BH-CCK-9 binding in presence of increasing concentrations of the non-hydrolyzable analog of GTP (GTP .S) on Cos cells membranes expressing the wild type and the mutated receptor reveales that the affinity of CCK-9 is not affected by GTPrS in the mutated receptor while in the wild type CCKBR the affinity of CCK-9 is strongly reduced. These results suggest that in the mutated receptor the coupling to the G protein that mediates PLC activation is strongly affected and may explain the lack of 1P production. These data should help to progress in the study of the molecular mechanisms involved in signal transduction that still remain poorly understood in the CCKBR. r
.
.
• G4682 CONSTITUTIVE ACTIVATION OF THE HUMAN VIP1/PACAP RECEPTOR, A MEMBER OF THE NEW CLASS II FAMILY OF G PROTEIN-COUPLED RECEPTORS. P. Gaudin, J.J. Maoret, A. Couvineau, C. Rouyer-Fessard, M. Laburthe, Paris, France.
The human VIPI/PACAP receptor belongs to the new class II subfamily of G protein-coupled receptors. We report here that the specific change by sitedirected mutagenesis of a strictly conserved histidine (H) into arginine (R) at position 178 of the human VIP1 receptor (junction between the first intracellular loop and second membrane-spanning domain) resulted in its constitutive, ligand-independent, activation with respect to cAMP production. Transfection of the H178R mutant into Cos cells resulted in a 3.5 foldincrease in the cAMP level as compared to cells transfected with the wild type receptor or the pcDNA3 vector alone. This increase was proportional to the amount of transfected eDNA encoding the H178R mutant. The H178R mutant exhibited an otherwise normal cAMP response to VIP in terms of potency and efficacy as well as a dissociation constant similar to that of the wild type receptor as determined by binding studies. Other mutants at position 178 such as H178K, H178A and H178D were not constitutively activated. They were otherwise expressed at the cell surface of transfected nonpermeabilized Cos ceils as assessed by immunofluorescence studies and antibody binding experiments carried out with receptors that were epitope-tagged in the N-terminal extracellular domain. Double mutants were then constructed in which the H178R mutation was associated with a point mutation in the the N-terminal extracellular domain that totally abolished VIP binding or VIP-stimulated cAMP production i.e. E36A or D68A. The corresponding double mutants H178R-E36A and HI78R-D68A were no longer constitutively activated. A control double mutant (HI78R-D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP, was still constitutively activated. Our findings demonstrate that constitutive activation of the VIPI/PACAP receptor by mutation of H178 into R requires the functional integrity of the N-terminal extracellular VIP binding domain. They might provide interesting generalities about the activation process in the class II family of G protein-coupled receptors. • G4683 A PROSPECTIVE STUDY OF GASTRIC LOCALIZATION IN PATIENTS WITH ZOLLINGER-ELLISON SYNDROME (ZES) WITH SOMATOSTATIN RECEPTOR SCINTIGRAPHY (SRS) AND ITS ASSOCIATION WITH THE PRESENCE OF GASTRIC CARCINOID TUMORS. F. Gibril, J.C. Reynolds, C.C. Chen, J. Serrano, S.U. Goebel, F. Yu, I.A. Lubensky, NIH, Bethesda, MD; J.A. Norton, UCSF Medical Center, San Francisco, CA; R.T. Jensen, NIH, Bethesda, MD.
Hypergastrinemic states cause an increase in the occurrence of gastric carcinoid tumors which can be difficult to identify. Recent studies show most carcinoid tumors and pancreatic endocrine tumors (PETs) have a high density of somatostatin receptors and that SRS is a very sensitive method to localize them. The purpose of the present study was to determine how frequently SRS localized to the stomach in patients with ZES and whether there was an association with the presence of gastric carcinoid tumors. 150 consecutive patients with ZES were studied prospectively with 32 having MEN-I, and 118 patients not having MEN-I. All patients underwent SRS with 6 mCi of [mIn-DTPA-DPhel]octreotide with SPECT imaging and upper
gastrointestinal endoscopy with Jumbo biopsies of any gastric abnormalities or if no abnormalities were seen two random biopsies of the gastric body were taken. Gastric carcinoid tumors were identified in 131150 (9%) of patients, 11/32 (34%) patients with MEN-I and 2/118 (2%) of patients without MEN-1. SRS demonstrated gastric localization in 121150 (8%) of patients, 10132 (31%) with MEN-I and 21118 (2%) of patients without MEN-I. In all 12 patients with positive gastric localization the localization was diffuse over the body of the stomach and in addition 4 of these patients had focal intense areas of localization. The focal intense localization was only seen in 4 patients with MEN-I. In the 32 patients with ZES and MEN-I, 9 of the I 1 (82%) patients with gastric carcinoid tumors had a positive SRS in the stomach. Of the 118 patients without MEN-I neither of the 2 patients with a gastric carcinoid had gastric uptake on the SRS and 2 of the 116 (2%) patients without MEN-I or a gastric carcinoid had uptake in the stomach. These results demonstrate that with SRS gastric localization is seen in 8% of ZES patients and occurs much more frequently (p <0.0001) in patients with MEN-I with ZES (31% vs 2%). Furthermore, gastric SRS localization was significantly (p<0.0001) associated with the presence of gastric carcinoid tumors (69% vs 2%). These results suggest SRS may be a useful method to localize gastric carcinoid tumors and conversely, it is important to remember in patients with ZES and/or MEN-I that SRS localization to the upper abdomen may represent a gastric carcinoid, not a primary or metastatic gastrinoma. G4684 AN AGGRESSIVE AND NONAGGRESSIVE FORM OF DISEASE EXISTS IN PATIENTS WITH MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 AND ZOLLINGER-ELLISON SYNDROME (ZES). F. Gibril, J. Serrano, F. Yu, S.U. Goebel, R.T. Jensen, NIH, Bethesda, MD.
Gastrinomas occur as a sporadic form (62-80%) and as a familial form with Multiple Endocrine Neoplasia-type 1 (MEN-I). A recent study shows that gastrinomas, in patients primarily with sporadic disease, can pursue an aggressive (25%) or nonaggressive form. In Older studies patients with MEN-I with ZES had a better prognosis and the tumor was thought to have an indolent course. However, a recent case report proposes aggressive forms of tumor growth may occur also in patients with MEN-I with ZES. The purpose of the present study was to determine whether an aggressive form of the disease occurs in patients with MEN-I, the percentage of MEN-I so affected, and whether these patients can be distinguished by laboratory or clinical parameters. All patients with MEN-I and ZES followed at the NIH were included. Patients underwent conventional imaging studies yearly (CT scan, ultrasound, MRI) and angiography if the conventional imaging results were unclear. In the last 3 years, yearly SRS was performed on all patients. If liver metastases were present patients had imaging studies every 3-6 months. Patients were divided into 2 groups, those with aggressive disease [rapid tumor growth (>25%/mo) which continued over subsequent admissions], or non aggressive disease. Patients had been followed from 4 months to 25 years. Five of 42 patients (12%) had aggressive tumor growth and 37142 (88%) did not have aggressive growth with a follow-up time of 8.4 and 7.6 years, respectively. Patients with and without aggressive disease did not differ in age, gender, duration of disease, basal acid output, percentage that underwent surgery or tumor location. Patients with aggressive disease more frequently had liver metastases (4/5=80% vs 3/37=8%, p=0.002), bone metastases (2/5=40% vs 0/37=0%, p=0.013) and had higher fasting gastrin levels (p <0.01). The difference in the rate of death from the gastrinoma [1/5 (20%) vs 0/31 (0%)] was not significant (p=0.11). These results demonstrate that in 12% of patients with MEN-I the gastrinoma pursues an aggressive course. These patients are significantly more likely to have liver, bone metastases and to have higher gastrin levels. At present patients with the aggressive form can not be distinguished from those without it unequivocally by any clinical or laboratory characteristic. Therefore, it will be important to compare these two groups in future studies for any differences in tumor markers or molecular abnormalities that will allow earlier anti-tumor strategies to be instituted in patients with aggressive disease. Furthermore, because a proportion of patients with ZES with MEN-I also have aggressive disease, these patients need to be followed carefully to attempt to detect this as soon as possible to institute antitumor treatment. G4685 POLYAMINES MODULATE PHAS-I GENE EXPRESSION IN AN INTESTINAL CELL LINE. John Gill, Karin Suess, ER Seidel. Department of Physiology, School of Medicine, East Carolina University, Greenville, NC. Ornithine decarboxylase (ODC), one of the rate limiting enzymes in polyamine biosynthesis, is a highly inducible enzyme whose activity is strictly regulated. Over-expression of ODC leads to neoplastic transformation. Regulation appears to posttranscriptional; ODC protein activity may increase 25-fold after growth factor stimulation with no change in steady state ODC mRNA levels. PHAS-I is a heat stable protein which may be a regulator of protein synthesis in response to agents which activate MAP kinases. PHAS-I or elF-4E binding protein limits the availability of elF-4E, a key component of the elF-4F translation initiation complex. Treatment of IEC-6 cells with PD 098059, a MEK 1 inhibitor, blocked the induction of ODC in repsonse to FBS by 90% confirming the potential involvement of MAP kinases in regulation of ODC translation. Cells were polyamine-depleted by 72 hr treatment with difluoromethylornithine, an inhibitor of ODC, followed by readdition of 10 uM putrescine. Total RNA was collected over the next 24 hr and ODC transcript level determined by ribonuclease protection assay. PHAS-I transcript levels began to fall 4 hr after putrescine and reached a nadir at 12 hr