TY - JOUR
T1 - Spatial Approximation between the Amino Terminus of a Peptide Agonist and the Top of the Sixth Transmembrane Segment of the Secretin Receptor
AU - Dong, Maoqing
AU - Li, Zhijun
AU - Pinon, Delia I.
AU - Lybrand, Terry P.
AU - Miller, Laurence J.
PY - 2004/1/23
Y1 - 2004/1/23
N2 - Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile p-benzoyl-L-phenylalanine into the position of His1 of rat secretin ([Bpa1,Tyr10]secretin-27). Because His1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions -1 (rat [Bpa-1,Tyr 10]secretin-27) and -2 (rat [Bpa-2, Gly -1,-Tyr10]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptor-bearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position -2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation.
AB - Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile p-benzoyl-L-phenylalanine into the position of His1 of rat secretin ([Bpa1,Tyr10]secretin-27). Because His1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions -1 (rat [Bpa-1,Tyr 10]secretin-27) and -2 (rat [Bpa-2, Gly -1,-Tyr10]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptor-bearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position -2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation.
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U2 - 10.1074/jbc.M310407200
DO - 10.1074/jbc.M310407200
M3 - Article
C2 - 14593094
AN - SCOPUS:1642494670
SN - 0021-9258
VL - 279
SP - 2894
EP - 2903
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -