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Comparative structural requirements of GRF analogs for occupancy and coupling to adenylate cyclase of GRF- and VIP receptors
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COMPARATIVE STRUCTURAL REQUIREMENTS OF GRF ANALOGS FOR OCCUPANCYAND COUPLING TO ADENYLATE CYCLASE OF GRF- AND VIP RECEPTORS . P.Robberecht,M. Waelbroeck, P.De Neef, J.C. Camus, J.Christophe and D. Coy÷. Dept. of Biochemistry and N u t r i t i o n , Faculty of Medicine, U.L.B. Brussels, Belgium and÷Deptof Medicine, Tulane University, New Orleans ,LO, USA . The a b i l i t y of synthetic analogs of GRF(1-29)NH2 to stimulate adenylate cyclase was investigated in a d e n o p i t u i t a r y - , l i v e r , and pancreatic membranes . In adenopituitary, GRF and analogs i n t e r a c t with GRF receptors only, whereas in l i v e r and pancreas they i n t e r a c t with VIP receptors . The GRF C-terminal moiety is required for GRF receptor recognition as synthetic helodermin,a parent peptide with 6 amino acids identical to those of GRF in the 1-10 sequence and the synthetic analog His -DAIa GRF(1-9)VIP(IO-28) stimulate p i t u i t a r y adenylate cyclase through occupation of VIP receptors only . When GRF analogs occupy GRF or VIP receptors, the N-terminal part of the molecule appears c r i t i c a l for subsequent adenylate cyclase a c t i v a t i o n . This was established by studying the e f f e c t of 30 analogs mono-,bi-, or trisubs~itute~ in po@ition@ 1 to~lO. ~ e major findings ~ere a~7the discfvery 9f Ac-~yr~-D~e , Dala~-DAsp~-DAsn~NLeu , His -DAla -DSer -Nleu and His~-DAla~-DThr'-Nleu ~" GRF(I-29)NHp as specific 1 antafonists of the VIP receptors of rat pancreas b) the discovery of Ac-Tyr DArg~GRF(I-29)NH2as an antagonist of p i t u i t a r y stimulated adenylate cyclase c)the importance of the free NHpradical on amino acid in position 1 for a c t i vation of adenylate cyclase through VIP receptors, but not through GRF receptors as acetylation of Tyr or His in position I decreases the i n t r i n s i c a c t i v i t y of the peptides in l i v e r and pancreatic membranes only. d) iodination of Tyr in position I decreases the potency and e f f i c a c y of GRF in a l l the systems tested .
INTERACTION OF SYNTHETIC N-AND C-TERMINAL FRAGMENTS OF HELODERMIN WITH RAT LIVER VIP RECEPTORS. P.Robberecht,N.Yanaihara~C.Yanaihara~,P.De Neef,J.C.Camus and J.Christophe Dept. of Biochemistry and N u t r i t i o n , F a c u l t y of Medicine,U.L.B., Brussels,Belgium and~Laboratory of Bioorganic Chemistry,Shizuoka College of Pharmacy,Shizuoka 422, Japan. Helodermin (Hd) is a 35 amino acid peptide isolated from Gila monster venom. I t s primary structure His-Ser-Asp-Ala-lle-Phe-Thr-Gln-Gln-Tyr-Ser-Lys-Leu-LeuA•a-Lys-Leu-A•a-Leu-G•n-Lys-Tyr-Leu-A•a-Ser-••e-Leu-G•y-Ser-Arg-Thr-Ser-Pr•-Pr•Pro-NH2 shows 15 amino acids identical to those of the VIP molecule (positions 1,2,3,~,6,7,10,13,18,21,22,23,25,26,27). The interaction of the synthetic peptide Hd(I-35)NH2, 1-27,7-35,13-35,17-35,18-35 and Hd(22-35)amide with rat l i v e r receptors was tested by i . the a b i l i t y of the peptides to i n h i b i t the binding of 1251-VIP,1251-Natural Hd and 1251Nd(l-35)NHp. 2. the a b i l i t y of the peptides to activate a membrane adenylate cyclase . A c~mparison with VIP,PHI,Secretin and synthetic fragments was performed . The results indicate that i . Hd(I-35)NHp stimulates e f f i c i e n t l y r a t hepatic adenylate cyclase through interaction wi~h the high a f f i n i t y VIP receptors (apparent Kact and Kd of 30 pM); the N-terminal fragment Hd(l-27)NHpis a f u l l agonist of lower potency (Kact and Kd of lOOpM) 2. The C-terminal f~agments Hd(7-35),13-35,17-35,and 18-35 amide stimulate also adenylate cyclase a c t i v i t y through occupation of VIP receptors with Kact and Kd of l , l , 3 , a n d IO~M respectively. The fragment Hd(22-35)NH 2 was inactive . 3. VIP fragments (ll-28)and (18-28),the PHI fragment (20-27) and the secretin fragment (7-27) are unable to i n t e r a c t with the rat hepatic receptor at a IO~M concentrat i o n . These results demonstrate that in rat l i v e r membranes both the N- and Cterminal parts of helodermin i n t e r a c t with the VIP receptor coupled to adenylate cyclase and suggest that the hydrophobic C-terminal part of the molecule although not d i r e c t l y involved in binding f a c i l i t a t e s the occupancy of the receptor.
COMPARATIVE STRUCTURAL REQUIREMENTS OF GRF ANALOGS FOR OCCUPANCYAND COUPLING TO ADENYLATE CYCLASE OF GRF- AND VIP RECEPTORS . P.Robberecht,M. Waelbroeck, P.De Neef, J.C. Camus, J.Christophe and D. Coy÷. Dept. of Biochemistry and N u t r i t i o n , Faculty of Medicine, U.L.B. Brussels, Belgium and÷Deptof Medicine, Tulane University, New Orleans ,LO, USA . The a b i l i t y of synthetic analogs of GRF(1-29)NH2 to stimulate adenylate cyclase was investigated in a d e n o p i t u i t a r y - , l i v e r , and pancreatic membranes . In adenopituitary, GRF and analogs i n t e r a c t with GRF receptors only, whereas in l i v e r and pancreas they i n t e r a c t with VIP receptors . The GRF C-terminal moiety is required for GRF receptor recognition as synthetic helodermin,a parent peptide with 6 amino acids identical to those of GRF in the 1-10 sequence and the synthetic analog His -DAIa GRF(1-9)VIP(IO-28) stimulate p i t u i t a r y adenylate cyclase through occupation of VIP receptors only . When GRF analogs occupy GRF or VIP receptors, the N-terminal part of the molecule appears c r i t i c a l for subsequent adenylate cyclase a c t i v a t i o n . This was established by studying the e f f e c t of 30 analogs mono-,bi-, or trisubs~itute~ in po@ition@ 1 to~lO. ~ e major findings ~ere a~7the discfvery 9f Ac-~yr~-D~e , Dala~-DAsp~-DAsn~NLeu , His -DAla -DSer -Nleu and His~-DAla~-DThr'-Nleu ~" GRF(I-29)NHp as specific 1 antafonists of the VIP receptors of rat pancreas b) the discovery of Ac-Tyr DArg~GRF(I-29)NH2as an antagonist of p i t u i t a r y stimulated adenylate cyclase c)the importance of the free NHpradical on amino acid in position 1 for a c t i vation of adenylate cyclase through VIP receptors, but not through GRF receptors as acetylation of Tyr or His in position I decreases the i n t r i n s i c a c t i v i t y of the peptides in l i v e r and pancreatic membranes only. d) iodination of Tyr in position I decreases the potency and e f f i c a c y of GRF in a l l the systems tested .
INTERACTION OF SYNTHETIC N-AND C-TERMINAL FRAGMENTS OF HELODERMIN WITH RAT LIVER VIP RECEPTORS. P.Robberecht,N.Yanaihara~C.Yanaihara~,P.De Neef,J.C.Camus and J.Christophe Dept. of Biochemistry and N u t r i t i o n , F a c u l t y of Medicine,U.L.B., Brussels,Belgium and~Laboratory of Bioorganic Chemistry,Shizuoka College of Pharmacy,Shizuoka 422, Japan. Helodermin (Hd) is a 35 amino acid peptide isolated from Gila monster venom. I t s primary structure His-Ser-Asp-Ala-lle-Phe-Thr-Gln-Gln-Tyr-Ser-Lys-Leu-LeuA•a-Lys-Leu-A•a-Leu-G•n-Lys-Tyr-Leu-A•a-Ser-••e-Leu-G•y-Ser-Arg-Thr-Ser-Pr•-Pr•Pro-NH2 shows 15 amino acids identical to those of the VIP molecule (positions 1,2,3,~,6,7,10,13,18,21,22,23,25,26,27). The interaction of the synthetic peptide Hd(I-35)NH2, 1-27,7-35,13-35,17-35,18-35 and Hd(22-35)amide with rat l i v e r receptors was tested by i . the a b i l i t y of the peptides to i n h i b i t the binding of 1251-VIP,1251-Natural Hd and 1251Nd(l-35)NHp. 2. the a b i l i t y of the peptides to activate a membrane adenylate cyclase . A c~mparison with VIP,PHI,Secretin and synthetic fragments was performed . The results indicate that i . Hd(I-35)NHp stimulates e f f i c i e n t l y r a t hepatic adenylate cyclase through interaction wi~h the high a f f i n i t y VIP receptors (apparent Kact and Kd of 30 pM); the N-terminal fragment Hd(l-27)NHpis a f u l l agonist of lower potency (Kact and Kd of lOOpM) 2. The C-terminal f~agments Hd(7-35),13-35,17-35,and 18-35 amide stimulate also adenylate cyclase a c t i v i t y through occupation of VIP receptors with Kact and Kd of l , l , 3 , a n d IO~M respectively. The fragment Hd(22-35)NH 2 was inactive . 3. VIP fragments (ll-28)and (18-28),the PHI fragment (20-27) and the secretin fragment (7-27) are unable to i n t e r a c t with the rat hepatic receptor at a IO~M concentrat i o n . These results demonstrate that in rat l i v e r membranes both the N- and Cterminal parts of helodermin i n t e r a c t with the VIP receptor coupled to adenylate cyclase and suggest that the hydrophobic C-terminal part of the molecule although not d i r e c t l y involved in binding f a c i l i t a t e s the occupancy of the receptor.