ABSTRACT
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.
Subject(s)
Ferrets/genetics , Losartan/metabolism , Receptors, Angiotensin/genetics , 1-Sarcosine-8-Isoleucine Angiotensin II/analogs & derivatives , 1-Sarcosine-8-Isoleucine Angiotensin II/metabolism , Alanine/genetics , Alanine/metabolism , Amino Acid Sequence , Amino Acid Substitution , Angiotensin II/analogs & derivatives , Angiotensin II/metabolism , Angiotensin II/pharmacology , Angiotensin Receptor Antagonists , Animals , Base Sequence , Binding Sites , Binding, Competitive/drug effects , COS Cells , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Recombinant/genetics , DNA, Recombinant/metabolism , Imidazoles/pharmacology , Iodine Radioisotopes , Losartan/pharmacology , Molecular Sequence Data , Plasmids , Protein Binding , Pyridines/pharmacology , Receptor, Angiotensin, Type 1 , Receptor, Angiotensin, Type 2 , Receptors, Angiotensin/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Threonine/genetics , Threonine/metabolismABSTRACT
The role of N-glycosylation in the pharmacological properties and cell surface expression of AT1 receptor was evaluated. Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putative N-linked glycosylation consensus sequences (N-X-S/T) of AT1 receptor (positions 4, 176, and 188) with aspartic acid. Expression of these mutant receptors in COS-7 cells followed by photolabeling with [125I]-[p-benzoyl-Phe8]AngII and SDS-PAGE revealed ligand-receptor complexes of four different molecular sizes, indicating that the three N-glycosylation sites are actually occupied by oligosaccharides. Binding studies showed that the affinity of each mutant receptor for [Sar1,Ile8]Ang II was not significantly different from that of wild-type AT1 receptor. Moreover, the functional properties of all mutant receptors were unaffected as evaluated by inositol phosphate production. However, the expression levels of the aglycosylated mutant were 5-fold lower than that of the wild-type AT1 receptor. Use of green fluorescent protein-AT1 receptor fusion proteins in studying the cellular location of the aglycosylated mutant demonstrated that it was distributed at a much higher density to the ER-Golgi complex than to the plasma membrane in HEK 293 cells. Together, these results suggest an important role of N-glycosylation in the proper trafficking of AT1 receptor to the plasma membrane.
