- Email: [email protected]
Transcriptional regulation of the TFF1 gene by gastrin
these from GRPR. The replacementof either EC2 or EC3 increasedaffinity for GRP [i.e. 2and 9-fold]. To determine the amino acid in the EC3 causing GRP selectivity, we replaced in the GRPR one at a time the 20 comparableEC3 amino acids that differed in the NMBR. Two separate NMBR substitutions in the GRPR, isoleucine (lie) for phenylalanine(Phe185)or lie for alanine (Ala198)altered GRP affinity decreasing by 25- and 16ofold, respectively. These results demonstratethat the selectivity nt the GRPRover the NMBR for GRP is doe entire~y to differences in the two receptor's 3'~ extracellular domain. Within this domain Phe~e~and Ala~ in the GRPR instead of isoleucine in the comparable positions in NMBR are the key amino acid differences determining GRP selectivity. These results suggest an interaction between aromatic ring of Phele5 with GRP possibly through amino-aromatic or aromaticaromatic interactions by cation-~ binding is likely important for high affinity GRP interaction. Furthermore, the decrease in affinity at position 198 in the GRPR by replacing alanine with isoleucine suggests either steric factors or hydrophobicity are important determinants at this site for GRP affinity.
535 Anchoring of Protein Kinase A by A-Kinase Anchoring Protein (AKAP) 150 Facilitates Pepsinogen Secretion from Gastric Chief Cells Guofeng Xie, Jean-Pierre Raufman, Univ of Arkansas for Medical Science, Little Rock, AR Previous data from our lab indicate the presence of cross-talk between signal transduction pathways and potentiation of pepsinogen secretion from gastric chief cells (JBC 271:19877,1996; BBA1357:73,1997). ThesepathwaysinvolvecAMP-dependentprotein kinase (PKA), protein kinase C (PKC) and calcineurin (CN). A family of proteins designatedAKAPs are reported to bind the regulatory subunit of PKA, PKC and CN and localize this protein complex to specific cell compartments. Objective:To determine whether an AKAP-anchored protein complex exists in chief ceils and whether this modulates secretion. Methods: In dispersed chief cells from guinea pig stomach: (a) To identify AKAPs and isoforms of PKA, PKC and CN, we preparedwestern blots from SDS-soluble extracts; (b) To identify proteins associatedwith AKAPs,we performed immunoprecipitationand affinity chromatographywith cAMP-agarosefrom NP-40-solubleextracts; (c) To determine subcellular distribution and colocalization of AKAPs and AKAP-associatedproteins, we used indirect immunofluorescent staining and contocal microscopy; and (d) To determine if association with AKAP 150 is necessaryfor full PKA activity, we examinedthe actions of Ht31, a peptide inhibitor of PKAAKAPassociation. Results: In western blots using two different antibodies,we identifiedAKAP 150, a rodent homolog of human AKAP 79. PKA I1~ or 11,8immunoprecipitated with AKAP 150. PKC,8II was also detectedin AKAP 150 immune- precipitates.Associationof PKA (~at) and CN was demonstrated by affinity chromatography. Confocal microscopy demonstrated colocalized staining at the cell periphery for AKAP 150 and PKC ,811.Addition of the Ht31 pegtide that competitively displaces PKA from the AKAP complex, but not an inactive Ht31P control peptide, inhibited pepsinogensecretion stimulated by a cAMP analogue,8-Br-cAMP. Ht31 did not inhibit secretion that was stimulated by agents whose actions are mediated by PKC and calcium (carbachol and A23187). Ht31, hut not Ht31P, inhibited carhachol- and A23187-stimuleted potentiationof secretionfrom cells preincubatedwith choleratoxin. Conclusions: In gastric chief cells, these data indicatethe existenceof a protein complexthat includes AKAP150, PKA,PKC/311,and CN. Disruption of the AKAP-PKAlinkageimpairs cAMP-mediated pepsinogen secretion and cross-talk between signal transduction pathways. AKAP 150, a scaffold protein, coordinates signaling events that mediate pepsinogensecretion.
533 Requirement for GoLqin COX-2 Expression by the GRP Receptor Lee Warren Slice, UCLA, Los Angeles, CA', Kyou-Bang Hart, Mel Simon, CA Institute of Technology, Pasadena,CA Background.We havedemonstratedthat gastrin releasingpeptide(GRP) inducescyclooxygenase-2 (COX-2) expressionthrough a Rho-dependentsignaling pathway. It is unclear whether Rho activation by the GRP receptor is through G
536 Identification of an Endogenous GLP-2 Receptor Antagonist Kazuhiro Watanabe, Elmi C. Tibaduiza,Ci Chen, New England Medical Ctr, Boston, MA; Yieh-Ping Wan, PerkinEImerLife Science, Boston, MA; Martin Beinborn, New England Medical Ctr, Boston, MA Glucagon-lika peptide 2 (GLP-2) is an intestinal hormone that induces mucosal growth and differentiation, as well as enhancesthe absorptivefunction of enterocytes.The GLP-2 receptor (GLP-2R) has been recently cloned and, based on amino acid homology, is classified as a member of the class B G-protein coupled receptor subfamily. Exploration of the GLP-2R's physiological role has been hampered by the paucity of pharmacologicaltools which would allow specific detection of receptor function and expression. Toward this goal, we have developeda novel radioligand where unmodified human GLP-2 was labeledat its free amino group with l~iodine by Boiton-Hunterconjugation. Homologouscompetition experimentswith this radioligand (50 pM) in COS-7 cells transiently expressing recombinant human GLP-2 receptor revealed a uniform population of high-affinity binding sites (Kd = 5 ni). Followup heterologouscompetition experiments indicatedthat the affinities of structurally related naturally occurring peptidss (e.g. glucagon-like peptide 1, glucagon, and lizard exendins) were at least 200-fold lower than that of GLP-2. In contrast, the primary endogenous metabolite of GLP-2 (resulting from enzymatic truncation of this 33 amino acid peptide by two amino terminal residues) maintainedan affinity comparableto that of the parent molecule (K, = 22 nU). Parallel assessment of GLP-2 receptor-mediatedsecond messenger signaling in COS7 cells revealedthat GLP-2 acted as an efficacious agonist of cAMP production (ECso= 0.4 nM) whereas amino-terminally truncated GLP-2 had only trace intrinsic activity (less than 10% of the full-length peptide). At the same time however,truncated GLP-2 maintainedhigh potency (ECso = 3 nM) consistent with its high receptor binding affinity. Further analysis revealedthat truncated GLP-2 acted as a competitive antagonist of full-length GLP-2 function, leadingto a concentration-dependentinhibition of agonist-stimulatedcAMP production. These findings indicate that systematic modification of GLP-2 at its amino terminus provides a promising strategytoward developinghigh affinity antagonists, useful both as pharmacological tools and as potential antiobesity drugs which act by reducing intestinal nutrient absorption. The observation that a primary metabolite of an endogenous receptor agonist can function as a near equipotent antagonist suggests a novel feed-back mechanism for fine-tuning the physiologic activity of gastrointestinal hormones.
534 Transcriptional Regulation of the TFF1 Gene by Gasfrin. Zara E. Khan, Univ of Liverpool, Liverpool United Kingdom; Timothy C. Wang, Univ of MA, Worcester, MA; Andrea Varro, Rod Dimaline, Univ of Liverpool, Liverpool United Kingdom Background: The antral hormone gastrin stimulates acid secretion and proliferation in the corpus. Studies in gastrin deficient transgenic mice (GAS-KO) suggest that gastrin also regulates corpus epithelial architecture. Aim: To identify genes regulated by gastrin using mRNA differential display. Methods: Genes expresseddifferentially in gastrin deficient (GASKO) and wild type (WT) mice were revealedby mRNAdifferential display. Changesin expression were confirmed and quantified by Northern analysis of gastric corpus from GAS-KO and hypergastrinaemictransgenic mice (iNS-GAS) versus their respectivecontrols. Acute regulation of geneexpressionwas studied in gastric adenocarcinoma-derivedcells (AGS)permanently transfected with the gastrin/CCKereceptor (AGSGR).Transcriptional regulation was assessed by reporter gene technology. Results: Differential display identified 18 genes down-regulated in GAS-KO mice, including that encoding the trefoil factor family member TFFI. Northern analysis confirmed that TFF1 mRNA was down-regulatedin the absence of gastrin (GAS-KO 63-7.0%-fold of WT, p
537
Examination Of Cholecysfokinin Receptor Dimerization Using Bioluminescence Resonance Energy Transfer Zhijie Cheng, LaurenceJ. Miller, Mayo Clin, Rochester, MN There has been increasing interest in the possibility of dimerization of G protein-coupled receptors,either through crossed-domaindimerizationor through the outside surface of intact helical bundles. We have recently reported that occupation of the CCK receptor by its natural ligand occurs as a one-to-one stoichiometric complex, eliminating crossed-domaindimerization of this receptor as a normal occurrence (J Med Chem 1999). In the current work, we have explored the possibility of the association of such complexes through their external surfaces using the method of bioluminescence resonance energy transfer (BRET). In this biophysical approach, we have examined potential interactions in intact cells between CCK receptor constructs that incorporated RenUlaluciferase(Rlu) and/or yellow fluorescent protein (YFP) at its carboxyl-terminal end. The positive control, incorporating both in series in a single molecule demonstrated a strong BRETsignal. In contrast, the signal observed when the two separate constructs were coexpressedin COS cells was ten percent of this control.
A-101
535 Anchoring of Protein Kinase A by A-Kinase Anchoring Protein (AKAP) 150 Facilitates Pepsinogen Secretion from Gastric Chief Cells Guofeng Xie, Jean-Pierre Raufman, Univ of Arkansas for Medical Science, Little Rock, AR Previous data from our lab indicate the presence of cross-talk between signal transduction pathways and potentiation of pepsinogen secretion from gastric chief cells (JBC 271:19877,1996; BBA1357:73,1997). ThesepathwaysinvolvecAMP-dependentprotein kinase (PKA), protein kinase C (PKC) and calcineurin (CN). A family of proteins designatedAKAPs are reported to bind the regulatory subunit of PKA, PKC and CN and localize this protein complex to specific cell compartments. Objective:To determine whether an AKAP-anchored protein complex exists in chief ceils and whether this modulates secretion. Methods: In dispersed chief cells from guinea pig stomach: (a) To identify AKAPs and isoforms of PKA, PKC and CN, we preparedwestern blots from SDS-soluble extracts; (b) To identify proteins associatedwith AKAPs,we performed immunoprecipitationand affinity chromatographywith cAMP-agarosefrom NP-40-solubleextracts; (c) To determine subcellular distribution and colocalization of AKAPs and AKAP-associatedproteins, we used indirect immunofluorescent staining and contocal microscopy; and (d) To determine if association with AKAP 150 is necessaryfor full PKA activity, we examinedthe actions of Ht31, a peptide inhibitor of PKAAKAPassociation. Results: In western blots using two different antibodies,we identifiedAKAP 150, a rodent homolog of human AKAP 79. PKA I1~ or 11,8immunoprecipitated with AKAP 150. PKC,8II was also detectedin AKAP 150 immune- precipitates.Associationof PKA (~at) and CN was demonstrated by affinity chromatography. Confocal microscopy demonstrated colocalized staining at the cell periphery for AKAP 150 and PKC ,811.Addition of the Ht31 pegtide that competitively displaces PKA from the AKAP complex, but not an inactive Ht31P control peptide, inhibited pepsinogensecretion stimulated by a cAMP analogue,8-Br-cAMP. Ht31 did not inhibit secretion that was stimulated by agents whose actions are mediated by PKC and calcium (carbachol and A23187). Ht31, hut not Ht31P, inhibited carhachol- and A23187-stimuleted potentiationof secretionfrom cells preincubatedwith choleratoxin. Conclusions: In gastric chief cells, these data indicatethe existenceof a protein complexthat includes AKAP150, PKA,PKC/311,and CN. Disruption of the AKAP-PKAlinkageimpairs cAMP-mediated pepsinogen secretion and cross-talk between signal transduction pathways. AKAP 150, a scaffold protein, coordinates signaling events that mediate pepsinogensecretion.
533 Requirement for GoLqin COX-2 Expression by the GRP Receptor Lee Warren Slice, UCLA, Los Angeles, CA', Kyou-Bang Hart, Mel Simon, CA Institute of Technology, Pasadena,CA Background.We havedemonstratedthat gastrin releasingpeptide(GRP) inducescyclooxygenase-2 (COX-2) expressionthrough a Rho-dependentsignaling pathway. It is unclear whether Rho activation by the GRP receptor is through G
536 Identification of an Endogenous GLP-2 Receptor Antagonist Kazuhiro Watanabe, Elmi C. Tibaduiza,Ci Chen, New England Medical Ctr, Boston, MA; Yieh-Ping Wan, PerkinEImerLife Science, Boston, MA; Martin Beinborn, New England Medical Ctr, Boston, MA Glucagon-lika peptide 2 (GLP-2) is an intestinal hormone that induces mucosal growth and differentiation, as well as enhancesthe absorptivefunction of enterocytes.The GLP-2 receptor (GLP-2R) has been recently cloned and, based on amino acid homology, is classified as a member of the class B G-protein coupled receptor subfamily. Exploration of the GLP-2R's physiological role has been hampered by the paucity of pharmacologicaltools which would allow specific detection of receptor function and expression. Toward this goal, we have developeda novel radioligand where unmodified human GLP-2 was labeledat its free amino group with l~iodine by Boiton-Hunterconjugation. Homologouscompetition experimentswith this radioligand (50 pM) in COS-7 cells transiently expressing recombinant human GLP-2 receptor revealed a uniform population of high-affinity binding sites (Kd = 5 ni). Followup heterologouscompetition experiments indicatedthat the affinities of structurally related naturally occurring peptidss (e.g. glucagon-like peptide 1, glucagon, and lizard exendins) were at least 200-fold lower than that of GLP-2. In contrast, the primary endogenous metabolite of GLP-2 (resulting from enzymatic truncation of this 33 amino acid peptide by two amino terminal residues) maintainedan affinity comparableto that of the parent molecule (K, = 22 nU). Parallel assessment of GLP-2 receptor-mediatedsecond messenger signaling in COS7 cells revealedthat GLP-2 acted as an efficacious agonist of cAMP production (ECso= 0.4 nM) whereas amino-terminally truncated GLP-2 had only trace intrinsic activity (less than 10% of the full-length peptide). At the same time however,truncated GLP-2 maintainedhigh potency (ECso = 3 nM) consistent with its high receptor binding affinity. Further analysis revealedthat truncated GLP-2 acted as a competitive antagonist of full-length GLP-2 function, leadingto a concentration-dependentinhibition of agonist-stimulatedcAMP production. These findings indicate that systematic modification of GLP-2 at its amino terminus provides a promising strategytoward developinghigh affinity antagonists, useful both as pharmacological tools and as potential antiobesity drugs which act by reducing intestinal nutrient absorption. The observation that a primary metabolite of an endogenous receptor agonist can function as a near equipotent antagonist suggests a novel feed-back mechanism for fine-tuning the physiologic activity of gastrointestinal hormones.
534 Transcriptional Regulation of the TFF1 Gene by Gasfrin. Zara E. Khan, Univ of Liverpool, Liverpool United Kingdom; Timothy C. Wang, Univ of MA, Worcester, MA; Andrea Varro, Rod Dimaline, Univ of Liverpool, Liverpool United Kingdom Background: The antral hormone gastrin stimulates acid secretion and proliferation in the corpus. Studies in gastrin deficient transgenic mice (GAS-KO) suggest that gastrin also regulates corpus epithelial architecture. Aim: To identify genes regulated by gastrin using mRNA differential display. Methods: Genes expresseddifferentially in gastrin deficient (GASKO) and wild type (WT) mice were revealedby mRNAdifferential display. Changesin expression were confirmed and quantified by Northern analysis of gastric corpus from GAS-KO and hypergastrinaemictransgenic mice (iNS-GAS) versus their respectivecontrols. Acute regulation of geneexpressionwas studied in gastric adenocarcinoma-derivedcells (AGS)permanently transfected with the gastrin/CCKereceptor (AGSGR).Transcriptional regulation was assessed by reporter gene technology. Results: Differential display identified 18 genes down-regulated in GAS-KO mice, including that encoding the trefoil factor family member TFFI. Northern analysis confirmed that TFF1 mRNA was down-regulatedin the absence of gastrin (GAS-KO 63-7.0%-fold of WT, p
537
Examination Of Cholecysfokinin Receptor Dimerization Using Bioluminescence Resonance Energy Transfer Zhijie Cheng, LaurenceJ. Miller, Mayo Clin, Rochester, MN There has been increasing interest in the possibility of dimerization of G protein-coupled receptors,either through crossed-domaindimerizationor through the outside surface of intact helical bundles. We have recently reported that occupation of the CCK receptor by its natural ligand occurs as a one-to-one stoichiometric complex, eliminating crossed-domaindimerization of this receptor as a normal occurrence (J Med Chem 1999). In the current work, we have explored the possibility of the association of such complexes through their external surfaces using the method of bioluminescence resonance energy transfer (BRET). In this biophysical approach, we have examined potential interactions in intact cells between CCK receptor constructs that incorporated RenUlaluciferase(Rlu) and/or yellow fluorescent protein (YFP) at its carboxyl-terminal end. The positive control, incorporating both in series in a single molecule demonstrated a strong BRETsignal. In contrast, the signal observed when the two separate constructs were coexpressedin COS cells was ten percent of this control.
A-101