Functional diversity of AT2 receptor orthologues in closely related species.
BACKGROUND
The most striking feature of life is biodiversity. However, mechanisms of biodiversity remain poorly understood, as most protein orthologues of different species are highly homologous in sequence and identical in function. Interestingly, recent evidence has demonstrated heterogeneity for a G protein-coupled angiotensin II (Ang II) type 2 (AT(2)) receptor in both ligand binding and induction of arachidonic acid (AA) release. The present study investigated the properties of AT(2) receptors in closely related species.


METHODS
AT(2) receptors cloned from human, rabbit, rat, and mouse were expressed in Chinese hamster ovary cells (CHO-K1), African green monkey kidney cells (COS-1), and human embryonic kidney (HEK)-293 cells and characterized in ligand binding and signal transductions. Critical residues in rabbit AT(2) receptor attributable to heterogeneity were examined using both gain-of-function and loss-of-function approaches with mutagenesis.


RESULTS
The newly cloned rabbit AT(2) receptor exhibits distinct biochemical and biologic properties compared to its highly homologous orthologues (91% in overall amino acid sequence) of rat, mouse, and human. All these orthologues activate SH2 domain-containing phosphatase-1 (SHP-1) and show similar binding affinities for Ang II and AT(2)-specific ligands CGP42112A and PD123319. However, reducing agent dithiothreitol (DTT) inactivates the rabbit orthologue but potentiates the others in ligand binding, a hallmark of AT(2) versus AT(1) receptor subtypes. Most interestingly, rabbit AT(2) receptor, but not the other orthologues, induces AA release in various cell systems when stimulated by both Ang II and CGP42112A, the peptide antagonist. Mutagenesis studies and sequence analyses further indicate that residues His(106), Asp(188), and Thr(293) are responsible for the DTT inactivation and residues Val(209) and Val(249) are partially responsible for AA release.


CONCLUSIONS
These results deny the coexistence of an additional AT(2) subtype in rabbit proximal tubule cells and demonstrate for the first time the presence of functional diversity for closely related Eutherian orthologues of a G protein-coupled receptor (GPCR) that are more than 90% homologous in the amino acid sequence.
Feng
Ying-Hong
YH
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA. yhfeng@usuhs.mil
Zhou
Lingyin
L
Sun
Yan
Y
Douglas
Janice G
JG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D044139:Receptor, Angiotensin, Type 2 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D044139:Receptor, Angiotensin, Type 2; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D054645:Protein Phosphatase 1; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C509248:Ptpn6 protein, mouse; C509250:Ptpn6 protein, rat; D004229:Dithiothreitol