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Identification of residues of the human CCK-A receptor that are in interaction with the c-terminal phenylalanine of CCK
April 2000
inhibitor (GF109203X, 100 nM, 20 min). In contrast, PKC activation had no effect on SP-induced Ca 2+ mobilization in cells expressing NKIRS324, NKIRS342, or KC4. PKC activation reduced, but did not abolish, responses in cells expressin.f NKIRS354 or KCl. Thus, activation of PKC abolishes SP-induced Ca 2 signalling in cells expressing wild type NKIR, suggesting that PKC plays a major role in NKIR desensitization. PKC may attenuate signalling by phosphorylating S338,T339 in the C-tail. Naturally occurring NKIRS324 is not regulated by PKC, which confirms that this receptor is differently regulated from the full length NKIR. Supported by DK39957. 1687 IDENTIFICATION OF RESIDUES OF THE HUMAN CCK-A RECEPTOR THAT ARE IN INTERACTION WITH THE C-TERMINAL PHENYLALANINE OF CCK. Chantal Escrieut, Bernard Maigret, Sandrine Sivente-Poirot, Veronique Gigoux, Jean Martinez, Luis Moroder, Eric Bignon, Nicole Vaysse, Daniel Fourmy, INSERM, Toulouse, France; CNRS Ua 510, Nancy, France; INSERM U151, Toulouse, France; CNRS UMR 5810, Montpellier, France; Max Planck Institute, Martinsried, Germany; Sanofi-Synthelabo, Toulouse, France. The cholecystokinin receptor-A (CCK-AR) mediates important cholecystokinin actions. The knowledge of its binding site represents important insights which may serve to understand its activation process and to design or optimize ligands. Previous studies have identified key aminoacids of the CCK-AR binding site for CCK and investigated their functional role (Kennedy et al., 1. Biol, Chern. 1997, 272, 2920-2926; Gigoux et al. in J. BioI. Chern. 1998,273, 14380-14386; 1999,274,20457-20464 and Prot. Sci., 1999, 8, 1-8). Further molecular-based docking of CCK into the experimentally validated 3-D model of the CCK-AR was carried out to identify aminoacids in interaction with the C-terminal Phe of CCK that is crucial for binding and biological activity. Four aromatic aminoacids (PheI70, Phe218, Trp326 and Phe330) were identified and individually mutated to Ala. Pharmacological characterization of the mutants was performed using transfected COS-7 cells and a battery of chemically distinct agonists (CCK9; JMV 180, SR 146,131) and antagonists( JMV 179, SR 27,897). Production of inositol phosphate was used as a functional test. All mutants bound the antagonist eHjSR 27,897 as the wild-type receptor. Competition binding to very low affinity state of the CCK-AR using [3H]SR 27,897 revealed that peptidic ligands (CCK-9, JMV 179 and 180) containing a Phe or a phenylethylester and SR 146,131, but not a CCK analogue lacking the Phe, bound with increased affinities (10 to 6O-fold) to mutants 218, 326 and 330. Mutants F218A and F330A bound CCK to a single class of binding sites with a lO-fold decreased affinity, and JMV 179 and 180 with a 5-fold increased affinity, respectively. Compared to the [WT]-CCK-AR, mutants 218, and 330 mediated CCK- and SR 146131stimulations of InsP with unchanged potencies but with 2-4 fold lower efficacies, while mutant 326 mediated CCK- and SR 146,13I-stimulations of insP with 30 and 5-fold lower potencies, respectively. These data are consistent with the conclusion that residues 218, 326 and 330 of the CCK-AR I) contribute to stabilize the CCK-AR in a resting state in absence of agonist 2) interact with the Phe of CCK and the phenyl group of SRI43,131, this interaction being important for CCK-AR transition to the high affinity PLC- coupled state. 1688 MUTATION OF ASN391 WITHIN THE HIGHLY CONSERVED NPXXY MOTIF OF THE CHOLECYSTOKININ B RECEPTOR ABOLISHES GQ PROTEIN ACTIVATION WITHOUT AFFECTING ITS ASSOCIATION WITH THE RECEPTOR. Celine Gales, Aline Kowalski-Chauvel, Marie-Noelle Dufour, Catherine Seva, Luis Moroder, Lucien Pradayrol, Nicole Vaysse, Daniel Fourmy, Sandrine Silvente-Poirot, INSERM U151, Toulouse, France; INSERM U469, Montpellier, France; Max Planck Institute, Martinsried, Germany. Highly conserved residues found in G-protein coupled receptors have been shown to be critical for maintaining normal receptor conformation and for other fundamental properties of the receptor such as high affinity binding, G protein coupling, signaling and regulation. Among the most conserved regions found in the G-protein coupled receptors is the (NID)PX2-3Y motif of the seventh transmembrane domain (where X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different
AGAA305
G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn391) of the NPXXY motif present in the CCKBR to Ala and determined the effects of the mutation on binding, signaling and G proteins coupling after transient expression of the mutated and wild type receptors in Cos cells. The mutated receptor displayed similar expression level and high affinity binding for CCK and selective agonists and antagonists compared to the wild-type CCKBR. However unlike the wild-type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase Codependent and independent mitogen activated protein kinases pathways, indicating an essential role of Asn391 in CCKBR signaling. Moreover, treatment with increasing concentrations of guanine-5'-O-(3-triotriphosphate) decreased the high affinity binding of CCK to the wild type receptor while no effect was observed with the mutated receptor, confirming the importance of Asn391 in G protein activation process. We used a combined immunoprecipitation and immunoblotting strategy to determine whether the inactive receptor mutant was associated to G proteins upon CCK stimulation. We showed that the inactive mutant retains an intact capacity to form stable complexes with G"q subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-Gq protein complexes is not sufficient to activate Gq proteins and suggest that Asn391 is specifically involved in Gq proteins activation. More generally, these results indicate that specific determinants of the receptor are involved in Gq activation while others are involved in Gq binding. Since this Asn residue is highly conserved in G-protein coupled receptors, it would be of interest to determine if a similar role could be extended to other receptors of this family. 1689 TOXICITY OF A POSSIBLE THERAPEUTIC GRF ANALOGUE IN VIVO IN MONKEYS BUT IN RATS IS LIKELY DUE TO VIP RECEPTOR AGONIST ACTIVITY. Hisato Igarashi, Tetsuhide Ito, Tapas K. Pradhan, Samuel A. Mantey, John E. Taylor, William A. Murphy, David H. Coy, Robert T. Jensen, NiddkNih, Bethesda, MD; Biomeasure Inc, Milford, MA; TULANE Med Sch, New Orleans, LA. Growth hormone-releasing factor (GRF ) is structurally related to vasoactive intestinal peptide and PACAP. Besides use in deficiency states, GH is used to treat short stature, aspects of aging, congestive heart failure and short bowel syndrome. Therefore, a stable GRF analogue could be therapeutically useful. Toward this aim, we svnthesized several analogues of GRF. The analogue [D-Ala 2, Aib 8 • 18, Ala~' 15. 16.22.24.26, Gab 27] hGRF(127)NH2 (GRF-A) had 7 times greater potency for stimulating GH release from rat dispersed pituitary cells, compared to human GRF(l-29)NH2 and had almost equal affinity for the human GRF-R. In toxicity studies, except for diarrhea, GRF-A had no effect on rats in high doses. However, when administrated intravenously to monkeys, they developed a syndrome within a few hours characterized by severe diarrhea and dehydration. In previous studies, some GRF analogues were found to interact with VIP receptors in some species. Therefore we considered the possibility GRF-A could be interacting with VIP-related receptors. To determine this, we studied its interaction with members of the VIP receptor family (PACAP-R, VIP1-R and VIP 2-R). We used rat VIPI receptor (rVIP1-R), human VIPI receptor (hVIP1-R), rVIP 2-R or hVIP 2-R stably transfected CHO and PANCI cells, pancreatic acini from rat or guinea pig which contain primarily VIP1-R and AR4-2J cells containing PACAP-R. For rat VIP receptors, hGRF(1-29)NH2 had low affinity (lC 50 > 750 nM). In contrast, VIP had high affinity for both rVIPeR and rVIP 2-R. GRF-A had low affinity for both rVIP I-R and rVIP2-R (> 1000 nM) and had low affinity for the PACAP receptor (> 3 JLM). For human VIP receptors, VIP had high affinity and hGRF(l-29)NH2 had low affinity (> 300 nM). In contrast GRF-A while having low affinity for hVIP 2-R (> 3 pM) had relatively high affinity for hVIP1-R (IC 5o - 66 oM). In guinea pig pancreatic acini, all peptides functioned as agonists at the VIP I-R causing enzyme secretion. These results demonstrate that in contrast to native hGRF(129)NH2, GRF-A has relatively higher affinity for the human VIP,-R but not for the human VIP 2-R, rat VIP-R or PACAP-R. These results raise the likely possibility that the observed effects in monkeys was due to the interaction of GRF-A with the VIP1-R. They demonstrate significant species differences exist for possible therapeutic peptide agonists and it is essential receptor affinity assessments be performed in human cells or closely related species.
inhibitor (GF109203X, 100 nM, 20 min). In contrast, PKC activation had no effect on SP-induced Ca 2+ mobilization in cells expressing NKIRS324, NKIRS342, or KC4. PKC activation reduced, but did not abolish, responses in cells expressin.f NKIRS354 or KCl. Thus, activation of PKC abolishes SP-induced Ca 2 signalling in cells expressing wild type NKIR, suggesting that PKC plays a major role in NKIR desensitization. PKC may attenuate signalling by phosphorylating S338,T339 in the C-tail. Naturally occurring NKIRS324 is not regulated by PKC, which confirms that this receptor is differently regulated from the full length NKIR. Supported by DK39957. 1687 IDENTIFICATION OF RESIDUES OF THE HUMAN CCK-A RECEPTOR THAT ARE IN INTERACTION WITH THE C-TERMINAL PHENYLALANINE OF CCK. Chantal Escrieut, Bernard Maigret, Sandrine Sivente-Poirot, Veronique Gigoux, Jean Martinez, Luis Moroder, Eric Bignon, Nicole Vaysse, Daniel Fourmy, INSERM, Toulouse, France; CNRS Ua 510, Nancy, France; INSERM U151, Toulouse, France; CNRS UMR 5810, Montpellier, France; Max Planck Institute, Martinsried, Germany; Sanofi-Synthelabo, Toulouse, France. The cholecystokinin receptor-A (CCK-AR) mediates important cholecystokinin actions. The knowledge of its binding site represents important insights which may serve to understand its activation process and to design or optimize ligands. Previous studies have identified key aminoacids of the CCK-AR binding site for CCK and investigated their functional role (Kennedy et al., 1. Biol, Chern. 1997, 272, 2920-2926; Gigoux et al. in J. BioI. Chern. 1998,273, 14380-14386; 1999,274,20457-20464 and Prot. Sci., 1999, 8, 1-8). Further molecular-based docking of CCK into the experimentally validated 3-D model of the CCK-AR was carried out to identify aminoacids in interaction with the C-terminal Phe of CCK that is crucial for binding and biological activity. Four aromatic aminoacids (PheI70, Phe218, Trp326 and Phe330) were identified and individually mutated to Ala. Pharmacological characterization of the mutants was performed using transfected COS-7 cells and a battery of chemically distinct agonists (CCK9; JMV 180, SR 146,131) and antagonists( JMV 179, SR 27,897). Production of inositol phosphate was used as a functional test. All mutants bound the antagonist eHjSR 27,897 as the wild-type receptor. Competition binding to very low affinity state of the CCK-AR using [3H]SR 27,897 revealed that peptidic ligands (CCK-9, JMV 179 and 180) containing a Phe or a phenylethylester and SR 146,131, but not a CCK analogue lacking the Phe, bound with increased affinities (10 to 6O-fold) to mutants 218, 326 and 330. Mutants F218A and F330A bound CCK to a single class of binding sites with a lO-fold decreased affinity, and JMV 179 and 180 with a 5-fold increased affinity, respectively. Compared to the [WT]-CCK-AR, mutants 218, and 330 mediated CCK- and SR 146131stimulations of InsP with unchanged potencies but with 2-4 fold lower efficacies, while mutant 326 mediated CCK- and SR 146,13I-stimulations of insP with 30 and 5-fold lower potencies, respectively. These data are consistent with the conclusion that residues 218, 326 and 330 of the CCK-AR I) contribute to stabilize the CCK-AR in a resting state in absence of agonist 2) interact with the Phe of CCK and the phenyl group of SRI43,131, this interaction being important for CCK-AR transition to the high affinity PLC- coupled state. 1688 MUTATION OF ASN391 WITHIN THE HIGHLY CONSERVED NPXXY MOTIF OF THE CHOLECYSTOKININ B RECEPTOR ABOLISHES GQ PROTEIN ACTIVATION WITHOUT AFFECTING ITS ASSOCIATION WITH THE RECEPTOR. Celine Gales, Aline Kowalski-Chauvel, Marie-Noelle Dufour, Catherine Seva, Luis Moroder, Lucien Pradayrol, Nicole Vaysse, Daniel Fourmy, Sandrine Silvente-Poirot, INSERM U151, Toulouse, France; INSERM U469, Montpellier, France; Max Planck Institute, Martinsried, Germany. Highly conserved residues found in G-protein coupled receptors have been shown to be critical for maintaining normal receptor conformation and for other fundamental properties of the receptor such as high affinity binding, G protein coupling, signaling and regulation. Among the most conserved regions found in the G-protein coupled receptors is the (NID)PX2-3Y motif of the seventh transmembrane domain (where X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different
AGAA305
G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn391) of the NPXXY motif present in the CCKBR to Ala and determined the effects of the mutation on binding, signaling and G proteins coupling after transient expression of the mutated and wild type receptors in Cos cells. The mutated receptor displayed similar expression level and high affinity binding for CCK and selective agonists and antagonists compared to the wild-type CCKBR. However unlike the wild-type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase Codependent and independent mitogen activated protein kinases pathways, indicating an essential role of Asn391 in CCKBR signaling. Moreover, treatment with increasing concentrations of guanine-5'-O-(3-triotriphosphate) decreased the high affinity binding of CCK to the wild type receptor while no effect was observed with the mutated receptor, confirming the importance of Asn391 in G protein activation process. We used a combined immunoprecipitation and immunoblotting strategy to determine whether the inactive receptor mutant was associated to G proteins upon CCK stimulation. We showed that the inactive mutant retains an intact capacity to form stable complexes with G"q subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-Gq protein complexes is not sufficient to activate Gq proteins and suggest that Asn391 is specifically involved in Gq proteins activation. More generally, these results indicate that specific determinants of the receptor are involved in Gq activation while others are involved in Gq binding. Since this Asn residue is highly conserved in G-protein coupled receptors, it would be of interest to determine if a similar role could be extended to other receptors of this family. 1689 TOXICITY OF A POSSIBLE THERAPEUTIC GRF ANALOGUE IN VIVO IN MONKEYS BUT IN RATS IS LIKELY DUE TO VIP RECEPTOR AGONIST ACTIVITY. Hisato Igarashi, Tetsuhide Ito, Tapas K. Pradhan, Samuel A. Mantey, John E. Taylor, William A. Murphy, David H. Coy, Robert T. Jensen, NiddkNih, Bethesda, MD; Biomeasure Inc, Milford, MA; TULANE Med Sch, New Orleans, LA. Growth hormone-releasing factor (GRF ) is structurally related to vasoactive intestinal peptide and PACAP. Besides use in deficiency states, GH is used to treat short stature, aspects of aging, congestive heart failure and short bowel syndrome. Therefore, a stable GRF analogue could be therapeutically useful. Toward this aim, we svnthesized several analogues of GRF. The analogue [D-Ala 2, Aib 8 • 18, Ala~' 15. 16.22.24.26, Gab 27] hGRF(127)NH2 (GRF-A) had 7 times greater potency for stimulating GH release from rat dispersed pituitary cells, compared to human GRF(l-29)NH2 and had almost equal affinity for the human GRF-R. In toxicity studies, except for diarrhea, GRF-A had no effect on rats in high doses. However, when administrated intravenously to monkeys, they developed a syndrome within a few hours characterized by severe diarrhea and dehydration. In previous studies, some GRF analogues were found to interact with VIP receptors in some species. Therefore we considered the possibility GRF-A could be interacting with VIP-related receptors. To determine this, we studied its interaction with members of the VIP receptor family (PACAP-R, VIP1-R and VIP 2-R). We used rat VIPI receptor (rVIP1-R), human VIPI receptor (hVIP1-R), rVIP 2-R or hVIP 2-R stably transfected CHO and PANCI cells, pancreatic acini from rat or guinea pig which contain primarily VIP1-R and AR4-2J cells containing PACAP-R. For rat VIP receptors, hGRF(1-29)NH2 had low affinity (lC 50 > 750 nM). In contrast, VIP had high affinity for both rVIPeR and rVIP 2-R. GRF-A had low affinity for both rVIP I-R and rVIP2-R (> 1000 nM) and had low affinity for the PACAP receptor (> 3 JLM). For human VIP receptors, VIP had high affinity and hGRF(l-29)NH2 had low affinity (> 300 nM). In contrast GRF-A while having low affinity for hVIP 2-R (> 3 pM) had relatively high affinity for hVIP1-R (IC 5o - 66 oM). In guinea pig pancreatic acini, all peptides functioned as agonists at the VIP I-R causing enzyme secretion. These results demonstrate that in contrast to native hGRF(129)NH2, GRF-A has relatively higher affinity for the human VIP,-R but not for the human VIP 2-R, rat VIP-R or PACAP-R. These results raise the likely possibility that the observed effects in monkeys was due to the interaction of GRF-A with the VIP1-R. They demonstrate significant species differences exist for possible therapeutic peptide agonists and it is essential receptor affinity assessments be performed in human cells or closely related species.