title: Topographical characterization of the domain structure of the bovine adrenal atrial natriuretic factor R1 receptor.
abstract: We have studied the structure and function of the membrane atrial natriuretic factor R1 (ANF-R1) receptor using limited proteolysis and exoglycosidase treatment. Limited digestion with trypsin of the receptor from bovine adrenal zona glomerulosa membranes resulted in the conversion of the native 130-kDa receptor into a single membrane-associated ANF-binding proteolytic fragment of 70 kDa. The 70-kDa fragment bound ANF with enhanced binding affinity but retained intact ANF-R1 pharmacological specificity and was still sensitive to modulation by amiloride. Trypsin treatment of the membranes produced a dual effect on ANF binding. Low concentrations of trypsin (less than or equal to 25 micrograms/mg of protein) increased ANF binding while higher concentrations dose dependently reduced the binding of the hormone. The increase of ANF-binding activity was associated with the formation of the 70-kDa fragment while the loss of ANF binding paralleled the degradation of the 70-kDa fragment. Low concentrations of trypsin drastically decreased the ANF-sensitive guanylate cyclase activity of the membrane fraction. This loss of catalytic activity strongly correlated with the formation of the 70-kDa tryptic fragment. We also evaluated the effect of ANF binding on the susceptibility of the receptor to proteolytic cleavage. The occupied receptor exhibited a greater sensitivity to trypsin digestion than the unoccupied protein, consistent with the hypothesis that hormone binding induces an important conformational change in the receptor structure. On the other hand, the 70-kDa fragment was much more resistant to proteolysis when occupied by ANF, suggesting that the ANF-binding domain forms a very compact structure.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Laboratory of Molecular Pharmacology, Clinical Research Institute of Montreal, Canada.
authors.forename: B
authors.initials: B
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Meloche
authors.forename: N
authors.initials: N
authors.lastname: McNicoll
authors.forename: C
authors.initials: C
authors.lastname: Lord
authors.forename: A
authors.initials: A
authors.lastname: De Léan
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D037102:Lectins / Q000494:pharmacology; D009439:Neuraminidase; D010446:Peptide Fragments / Q000378:metabolism; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D014357:Trypsin; D015384:Zona Glomerulosa / Q000378:metabolism*
chemical_list: D037102:Lectins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D000584:Amiloride; D009320:Atrial Natriuretic Factor; D009439:Neuraminidase; D014357:Trypsin; D017461:Receptors, Atrial Natriuretic Factor