Highly efficient photoaffinity labeling of the hormone binding domain of atrial natriuretic factor receptor.
A high-efficiency photoaffinity derivative of atrial natriuretic factor (ANF) was developed for studying the peptide binding domain of the receptor protein and for better characterization of this receptor in tissues with a low density of binding sites. The position of the photosensitive residue was chosen on the basis of a molecular conformational model and on structure-activity relationship studies which both indicate that the carboxy-terminal end of the peptide is part of a hydrophobic pole likely to interact deeply within the ANF binding pocket of the receptor. Selection of the photoreactive residue p-benzoylphenylalanine (BPA) as a substitute for arginine in position 125 of the peptide sequence led to a photoaffinity derivative with a high (63%) efficiency of covalent incorporation to the receptor protein. This derivative (BPA-ANF) has a 10-fold lower affinity when compared with ANF, but it is a full agonist in stimulating cGMP production and inhibiting aldosterone secretion in bovine adrenal zona glomerulosa. Photoaffinity labeling with BPA-ANF specifically identifies ANF-R1 and ANF-R2 receptor proteins with a 10-fold higher efficiency than with azido derivatives of ANF or with cross-linking agents. This new ANF derivative therefore appears to be useful for studying ANF receptors in tissues with low levels of expression, for locating receptor following cellular internalization, and for tagging proteolytic fragments of the receptor amenable to amino acid microsequencing.
McNicoll
N
N
Clinical Research Institute of Montreal, Canada.
Escher
E
E
Wilkes
B C
BC
Schiller
P W
PW
Ong
H
H
De Léan
A
A
D000345:Affinity Labels; D000450:Aldosterone / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000138:chemical synthesis / Q000737:chemistry*; D002417:Cattle; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D010777:Photochemistry; D011485:Protein Binding; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000737:chemistry*
D000345:Affinity Labels; D010455:Peptides; D011956:Receptors, Cell Surface; D010649:Phenylalanine; D000450:Aldosterone; D009320:Atrial Natriuretic Factor; C053764:benzoylphenylalanine; D017461:Receptors, Atrial Natriuretic Factor