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Characterization of a constitutively active mutant of the human vasoactive intestinal peptide (VIP) 1 receptor
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Abstracts from the l l th International Symposium on Regulatory Peptides
D I S T R I B U T I O N OF N E U R O P E P T I D E Y - I M M U N O R E A C T I V E NERVES IN HEPATOLITHIASIS Masaki Fujimura, Takashi Kinoshita, Ikuo Y a m a m o t o Second Department of Surgery, Shiga University of Medical Science, Seta, Otsu, Shiga 520-21 Japan Hepatolithiasis, or intrahepatic stone, is a c o m m o n disease in Southeast Asia. In the present study, the distribution of neuropeptide Y (NPY)-immunoreactive nerves in the human liver with hepatolithiasis was studied using immunohistochemistry. Liver specimens including stenotic portions of the bile duct were taken at operation from 19 Japanese patients. In the intact portion of the liver, NPY-immunoreactive nerve fibers were densely distributed in the Glisson's sheath, where nerve fibers were most abundant in the perivascular portion, and were also observed in the wall of vessels and bile ducts.
The distribution pattern of NPY-
immunoreactive nerve fibers changed according to the degree of cholangitis.
In specimens
with lymphocyte infiltration, a reduction or disappearance of N P Y - i m m u n o r e a c t i v e nerve fibers was observed in the wall and around the bile duct, whereas not so many changes were found in specimens of fibrotic bile duct with glandular proliferation. On the other hand, positive nerve fibers in the perivascular plexus were well preserved, independent of the degree of cholangitis. Although the physiological significance is not known, the unique distribution of N P Y - i m m u n o r e a c t i v e nerves shown in this study suggests some relationship with the etiology of hepatolithiasis.
CHARACTERIZATION OF A CONSTITUTIVELY ACTIVE MUTANT OF THE HUMAN VASOACTIVE INTESTINAL PEPTIDE (VIP) 1 RECEPTOR Paseale Gaudin, Christiane Rouyer-Fessard, Ahin Couvineau, Jean-Jos6 Maoret and Marc Laburthe. INSERM U410, Neuroendocrinology and Cell Biology of the Digestive System, Facult~ de Medecine Xavier Bichat, B.P. 416, 75870 Paris Cedex, France The human VIP 1 receptor belongs to the new subfamily of G protein-coupled receptors that includes secretin, glucagon, PACAP, GLP-1, GIP, PTH and calcitonin receptors. We report here that the specific change of a strictly conserved histidine (H) residue to argmine (R) at position 178 of the human VIP 1 receptor (junction between the first intracellular loop and second membrane-spenning domain) resulted in its constitutive, ligand-independent activation. The HI78R receptor mutant was obtained by site-directed mutagenesis and the mutated cDNA inserted into the pcDNA3 vector was transfected into Cos cells (Monkey kidney cells). This results in a 4-5 fold-increase in the cAMP levels in Cos cells as compared to cells transfected with the wild-type cDNA or vector alone. Similar data were obtained upon transfection of CHO cells (Chinese hamster ovary). The increase in cAMP level in Cos cells transfected with the HI78K mutant is proportional to the amount of tranfected plasmid. Although the HI 78R receptor was constitutively activated, it still mediated VIP-stimulated cAMP levels in Cos cells with a maximal effect similar to that observed with the wild-type receptor. Binding studies in Cos cells transfected with the H178R and wild-type receptor indicated a Kd = 0.20~0.08 nM and 0.62-4:0.05 nM and a Bmax = 1.97:L-0.93 and 6.10-a:l.5l pmoles/mg protein, respectively. Other mutants were also generated at position 178 by changing histidine (H) to lysine (K), alanine (A) or aspartate (D). The H178K, HI78A and H178D receptors did not bind VIP and did not mediate VIP-stimulated cAMP production when transfected into Cos cells. Since immunofluorescence studies showed that these latter mutants were normally delivered at the plasma membrane, they were probably inactive. It is concluded that i) a highly specific change at position 178 (H to R) of the human VIPI receptor results in a constitutive activation of this receptor; ii) position 178 is critical for VIP receptor activity. Whether such mutation occurs in human pathology is yet unknown but mutation of this conserved histine at position 223 of the human PTH receptor was previously reported to be associated with constitutive activation of the PTH receptor in Jansen-type metaphyseal chondrodysplasia.
Abstracts from the l l th International Symposium on Regulatory Peptides
D I S T R I B U T I O N OF N E U R O P E P T I D E Y - I M M U N O R E A C T I V E NERVES IN HEPATOLITHIASIS Masaki Fujimura, Takashi Kinoshita, Ikuo Y a m a m o t o Second Department of Surgery, Shiga University of Medical Science, Seta, Otsu, Shiga 520-21 Japan Hepatolithiasis, or intrahepatic stone, is a c o m m o n disease in Southeast Asia. In the present study, the distribution of neuropeptide Y (NPY)-immunoreactive nerves in the human liver with hepatolithiasis was studied using immunohistochemistry. Liver specimens including stenotic portions of the bile duct were taken at operation from 19 Japanese patients. In the intact portion of the liver, NPY-immunoreactive nerve fibers were densely distributed in the Glisson's sheath, where nerve fibers were most abundant in the perivascular portion, and were also observed in the wall of vessels and bile ducts.
The distribution pattern of NPY-
immunoreactive nerve fibers changed according to the degree of cholangitis.
In specimens
with lymphocyte infiltration, a reduction or disappearance of N P Y - i m m u n o r e a c t i v e nerve fibers was observed in the wall and around the bile duct, whereas not so many changes were found in specimens of fibrotic bile duct with glandular proliferation. On the other hand, positive nerve fibers in the perivascular plexus were well preserved, independent of the degree of cholangitis. Although the physiological significance is not known, the unique distribution of N P Y - i m m u n o r e a c t i v e nerves shown in this study suggests some relationship with the etiology of hepatolithiasis.
CHARACTERIZATION OF A CONSTITUTIVELY ACTIVE MUTANT OF THE HUMAN VASOACTIVE INTESTINAL PEPTIDE (VIP) 1 RECEPTOR Paseale Gaudin, Christiane Rouyer-Fessard, Ahin Couvineau, Jean-Jos6 Maoret and Marc Laburthe. INSERM U410, Neuroendocrinology and Cell Biology of the Digestive System, Facult~ de Medecine Xavier Bichat, B.P. 416, 75870 Paris Cedex, France The human VIP 1 receptor belongs to the new subfamily of G protein-coupled receptors that includes secretin, glucagon, PACAP, GLP-1, GIP, PTH and calcitonin receptors. We report here that the specific change of a strictly conserved histidine (H) residue to argmine (R) at position 178 of the human VIP 1 receptor (junction between the first intracellular loop and second membrane-spenning domain) resulted in its constitutive, ligand-independent activation. The HI78R receptor mutant was obtained by site-directed mutagenesis and the mutated cDNA inserted into the pcDNA3 vector was transfected into Cos cells (Monkey kidney cells). This results in a 4-5 fold-increase in the cAMP levels in Cos cells as compared to cells transfected with the wild-type cDNA or vector alone. Similar data were obtained upon transfection of CHO cells (Chinese hamster ovary). The increase in cAMP level in Cos cells transfected with the HI78K mutant is proportional to the amount of tranfected plasmid. Although the HI 78R receptor was constitutively activated, it still mediated VIP-stimulated cAMP levels in Cos cells with a maximal effect similar to that observed with the wild-type receptor. Binding studies in Cos cells transfected with the H178R and wild-type receptor indicated a Kd = 0.20~0.08 nM and 0.62-4:0.05 nM and a Bmax = 1.97:L-0.93 and 6.10-a:l.5l pmoles/mg protein, respectively. Other mutants were also generated at position 178 by changing histidine (H) to lysine (K), alanine (A) or aspartate (D). The H178K, HI78A and H178D receptors did not bind VIP and did not mediate VIP-stimulated cAMP production when transfected into Cos cells. Since immunofluorescence studies showed that these latter mutants were normally delivered at the plasma membrane, they were probably inactive. It is concluded that i) a highly specific change at position 178 (H to R) of the human VIPI receptor results in a constitutive activation of this receptor; ii) position 178 is critical for VIP receptor activity. Whether such mutation occurs in human pathology is yet unknown but mutation of this conserved histine at position 223 of the human PTH receptor was previously reported to be associated with constitutive activation of the PTH receptor in Jansen-type metaphyseal chondrodysplasia.