Deglycosylation and fragmentation of purified rat liver angiotensin II receptor: application to the mapping of hormone-binding domains.
We report new structural data about the rat liver angiotensin II receptor, which belongs to the AT1 subclass. This receptor has been purified at analytical or semi-preparative levels by a previously described strategy involving its photolabelling with a biotinylated azido probe and selective adsorption of the covalent probe-receptor complexes to immobilized streptavidin [Marie, Seyer, Lombard, Desarnaud, Aumelas, Jard and Bonnafous (1990) Biochemistry 29, 8943-8950]. Chemical or enzymic deglycosylation of the purified receptor has shown a shift in its molecular mass from 65 kDa to 40 kDa. Fragmentation of the purified receptor was carried out with V8 protease from Staphylococcus aureus, CNBr and trypsin. It was possible to find trypsin-treatment conditions which allowed production of a 6 kDa probe-fragment complex with a satisfactory yield. Attempts to localize this small fragment (5 kDa after subtraction of the probe contribution) in the recently published rat AT1 receptor sequence are reported. As expected, this fragment is not glycosylated; moreover, its further fragmentation by CNBr induces a very slight decrease in its size. These data support the hypothesis that a receptor sequence comprising the third transmembrane domain and adjacent portions of extra- and intracellular loops is involved in photolabelling by the C-terminal azidophenylalanine of the angiotensin-derived probe. These preliminary results are discussed in terms of future prospects for the characterization of hormone-binding domains of angiotensin II receptors.
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.
F
F
Desarnaud
J
J
Marie
C
C
Lombard
R
R
Larguier
R
R
Seyer
T
T
Lorca
S
S
Jard
J C
JC
Bonnafous
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011945:Receptors, Angiotensin / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D012697:Serine Endopeptidases; D014357:Trypsin
D010446:Peptide Fragments; D011945:Receptors, Angiotensin; D011956:Receptors, Cell Surface; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D003488:Cyanogen Bromide