Molecular cloning of a ferret angiotensin II AT(1) receptor reveals the importance of position 163 for Losartan binding.
A complementary DNA for the angiotensin II (AngII) type 1 (AT(1)) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT(1)) of 359 amino acids having high homologies (93-99%) to other mammalian AT(1) receptor counterparts. When fAT(1) was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue (125)I-¿Sar(1), Bpa(8)AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT(1) non-peptidic antagonist L-158,809. Functional analysis indicated that the fAT(1) receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT(1) receptor had a high affinity for the peptide antagonist ¿Sar(1), Ile(8)AngII (K(d) of 5. 8+/-1.4 nM) but a low affinity for the AT(1) selective non-peptidic antagonist DuP 753 (K(d) of 91+/-15.6 nM). Interestingly, when we substituted Thr(163) with an Ala residue, which occupies this position in many mammalian AT(1) receptors, we restored the high affinity of this receptor for Dup 753 (11.7+/-5.13 nM). These results suggest that position 163 of the AT(1) receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.
Gosselin
M J
MJ
Department of Pharmacology, Faculty of Medicine, Universit¿e de Sherbrooke, Quebec, J1H 5N4, Sherbrooke, Canada.
Leclerc
P C
PC
Auger-Messier
M
M
Guillemette
G
G
Escher
E
E
Leduc
R
R
D015059:1-Sarcosine-8-Isoleucine Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D057911:Angiotensin Receptor Antagonists; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D005289:Ferrets / Q000235:genetics*; D007093:Imidazoles / Q000494:pharmacology; D007457:Iodine Radioisotopes; D019808:Losartan / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D057911:Angiotensin Receptor Antagonists; D018076:DNA, Complementary; D004274:DNA, Recombinant; D007093:Imidazoles; D007457:Iodine Radioisotopes; D011725:Pyridines; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; C099368:angiotensin II, iodo-(Sar(1)-Ile(8))-; D000804:Angiotensin II; C073402:PD 123319; D013912:Threonine; D015059:1-Sarcosine-8-Isoleucine Angiotensin II; D019808:Losartan; D000409:Alanine