Expression and purification of the extracellular ligand-binding domain of the atrial natriuretic peptide (ANP) receptor: Monovalent binding with ANP induces 2 : 2 complexes
Ks. Misono et al., Expression and purification of the extracellular ligand-binding domain of the atrial natriuretic peptide (ANP) receptor: Monovalent binding with ANP induces 2 : 2 complexes, BIOCHEM, 38(2), 1999, pp. 516-523
The receptor for atrial natriuretic peptide (ANP) is a type-I transmembrane
protein containing an extracellular ligand-binding domain, a single transm
embrane sequence, an intracellular kinase-homologous domain, and a guanylat
e cyclase (GCase) domain. Binding of ANP to the extracellular domain causes
activation of the GCase domain by an as yet unknown mechanism. To facilita
te studies of the receptor structure and signaling mechanism, we have expre
ssed the extracellular ANP-binding domain of rat ANP receptor (NPR-ECD) in
a water-soluble form. NPR-ECD was purified to homogeneity by ANP-affinity c
hromatography. SDS-PAGE gave a single 61-kDa band, which coincided with a r
adioactive band obtained by photoaffinity-labeling with N-4 alpha-azidobenz
oyl-I-125-ANP(4-28). Edman degradation gave a single amino-terminal sequenc
e expected for the mature protein. Both trifluoromethanesulfonic acid and p
eptide-N-glycosidase F treatments yielded a 50-kDa band, indicating N-glyco
sylation. The molecular mass of 57 725 Da determined by mass spectrometry i
ndicates the carbohydrate content at 16%. NPR-ECD bound ANP with an affinit
y comparable to that of the full-length receptor. The ligand selectivity of
NPR-ECD (in the order ANP > brain natriuretic peptide >> C-type natriureti
c peptide) was also similar to that of the full-length receptor. HPLC gel f
iltration of NPR-ECD gave a peak with an apparent mass of 74 kDa, Preincuba
tion with ANP generated a new 150-kDa peak with a concomitant decrease of t
he 74-kDa peak. This shift in peak positions was ANP concentration-dependen
t and was complete at the NPR-ECD-to-ANP molar ratio of 1:I, indicating equ
imolar binding. The change in the apparent native molecular weight from 74
to 150 kDa suggests that binding causes dimerization of the NPR-ECD:ANP com
plex to yield an [NPR-ECD:ANP](2) complex.