TY - JOUR
T1 - Demonstration of a direct interaction between residue 22 in the carboxyl-terminal half of secretin and the amino-terminal tail of the secretin receptor using photoaffinity labeling
AU - Dong, Maoqing
AU - Wang, Yan
AU - Pinon, Delia I.
AU - Hadac, Elizabeth M.
AU - Miller, Laurence J.
PY - 1999/1/8
Y1 - 1999/1/8
N2 - An understanding of the molecular basis of hormonal activation of receptors provides important insights for drug design. Toward this end, intrinsic photoaffinity labeling is a powerful tool to directly identify the ligand-binding domain. We have developed a new radioiodinatable agonist ligand of the secretin receptor that incorporates a photolabile p-benzoyl-L- phenylalanine (Bpa) into the position of Leu22 and have utilized this to identify the adjacent receptor domain. The rat [Tyr10,Bpa22]secretin-27 probe was a fully efficacious agonist, with a potency to stimulate cAMP accumulation by Chinese hamster ovary SecR cells similar to that of natural secretin (EC50 = 68 ± 22 pM analogue and 95 ± 25 pM secretin). It bound specifically and with high affinity (K(i) = 5.0 ± 1.1 nM) and covalently labeled the M(r) = 57,000-62,000 secretin receptor. Cyanogen bromide cleavage of the receptor yielded a major labeled fragment of apparent M(r) = 19,000 that shifted to M(r) = 9,000 after deglycosylation. This was most consistent with either of two glycosylated domains within the amino-terminal tail of the receptor. Immunoprecipitation with antibody directed to epitope tags incorporated into each of the candidate domains established that the fragment at the amino terminus of the receptor was the site of labeling. This was further localized to the amino-terminal 30 residues of the receptor by additional proteolysis of this fragment with endoproteinase Lys-C. This provides the first direct demonstration of a contact between a secretin-like agonist and its receptor and will contribute a useful constraint to the modeling of this interaction.
AB - An understanding of the molecular basis of hormonal activation of receptors provides important insights for drug design. Toward this end, intrinsic photoaffinity labeling is a powerful tool to directly identify the ligand-binding domain. We have developed a new radioiodinatable agonist ligand of the secretin receptor that incorporates a photolabile p-benzoyl-L- phenylalanine (Bpa) into the position of Leu22 and have utilized this to identify the adjacent receptor domain. The rat [Tyr10,Bpa22]secretin-27 probe was a fully efficacious agonist, with a potency to stimulate cAMP accumulation by Chinese hamster ovary SecR cells similar to that of natural secretin (EC50 = 68 ± 22 pM analogue and 95 ± 25 pM secretin). It bound specifically and with high affinity (K(i) = 5.0 ± 1.1 nM) and covalently labeled the M(r) = 57,000-62,000 secretin receptor. Cyanogen bromide cleavage of the receptor yielded a major labeled fragment of apparent M(r) = 19,000 that shifted to M(r) = 9,000 after deglycosylation. This was most consistent with either of two glycosylated domains within the amino-terminal tail of the receptor. Immunoprecipitation with antibody directed to epitope tags incorporated into each of the candidate domains established that the fragment at the amino terminus of the receptor was the site of labeling. This was further localized to the amino-terminal 30 residues of the receptor by additional proteolysis of this fragment with endoproteinase Lys-C. This provides the first direct demonstration of a contact between a secretin-like agonist and its receptor and will contribute a useful constraint to the modeling of this interaction.
UR - http://www.scopus.com/inward/record.url?scp=0033534683&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033534683&partnerID=8YFLogxK
U2 - 10.1074/jbc.274.2.903
DO - 10.1074/jbc.274.2.903
M3 - Article
C2 - 9873030
AN - SCOPUS:0033534683
SN - 0021-9258
VL - 274
SP - 903
EP - 909
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 2
ER -