Structural Basis of G Protein Specificity of Human Endothelin Receptors

Abstract

The endothelin (ET) family of peptides acts via two subtypes of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors termed ETAand ETB. ET-1 stimulated cAMP formation in Chinese hamster ovary (CHO) cells stably expressing human wild-type ETA(CHO/hETAcells) while it inhibited cAMP formation in CHO cells expressing human wild-type ETB(CHO/hETBcells), and pharmacological evidence indicated that the opposite effects were due to the selective coupling of each receptor subtype with Gαs/Gαi. To find out a receptor domain(s) that determined the selective coupling, a series of chimeric receptors between hETAand hETBwas expressed on CHO cells, and the effect of ET-1 on cAMP formation in each cell line was tested. hETAwith the replacement of second and/or third intracellular loop (ICLII and/or -III) to the corresponding region(s) of hETBfailed to transmit the stimulatory effect of ET-1. hETBwith the replacement of ICLIII to the corresponding region of hETAfailed to transmit the inhibitory effect of ET-1. A chimeric receptor with ICLII of hETBand with ICLIII of hETAfailed to transmit both effects. In cells expressing chimeric receptors with ICLII of hETAand with ICLIII of hETB, ET-1 inhibited cAMP formation while it stimulated cAMP formation when cells were pretreated with pertussis toxin. These results indicated the roles of ICLII and -III of hETR as a major determinant of the selective coupling of hETAand hETBwith Gαs/Gαi, respectively. We also demonstrated that each receptor subtype expressed on the same cell could work independently, i.e. for hETAto activate Gαs and for hETBto activate Gαi, resulting in dose-dependent dual effects of ET-1 on cAMP formation. The endothelin (ET) family of peptides acts via two subtypes of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors termed ETAand ETB. ET-1 stimulated cAMP formation in Chinese hamster ovary (CHO) cells stably expressing human wild-type ETA(CHO/hETAcells) while it inhibited cAMP formation in CHO cells expressing human wild-type ETB(CHO/hETBcells), and pharmacological evidence indicated that the opposite effects were due to the selective coupling of each receptor subtype with Gαs/Gαi. To find out a receptor domain(s) that determined the selective coupling, a series of chimeric receptors between hETAand hETBwas expressed on CHO cells, and the effect of ET-1 on cAMP formation in each cell line was tested. hETAwith the replacement of second and/or third intracellular loop (ICLII and/or -III) to the corresponding region(s) of hETBfailed to transmit the stimulatory effect of ET-1. hETBwith the replacement of ICLIII to the corresponding region of hETAfailed to transmit the inhibitory effect of ET-1. A chimeric receptor with ICLII of hETBand with ICLIII of hETAfailed to transmit both effects. In cells expressing chimeric receptors with ICLII of hETAand with ICLIII of hETB, ET-1 inhibited cAMP formation while it stimulated cAMP formation when cells were pretreated with pertussis toxin. These results indicated the roles of ICLII and -III of hETR as a major determinant of the selective coupling of hETAand hETBwith Gαs/Gαi, respectively. We also demonstrated that each receptor subtype expressed on the same cell could work independently, i.e. for hETAto activate Gαs and for hETBto activate Gαi, resulting in dose-dependent dual effects of ET-1 on cAMP formation.