PAR1 thrombin receptor-G protein interactions. Separation of binding and coupling determinants in the galpha subunit.
Signal transfer between the protease-activated PAR1 thrombin receptor and membrane-associated heterotrimeric G proteins is mediated by protein-protein interactions. We constructed a yeast signaling system that resolves domain-specific functions of binding from coupling in the Galpha subunit. The endogenous yeast Galpha subunit, Gpa1, does not bind to PAR1 and served as a null structural template. N- and C-terminal portions of mammalian G(i2) and G(16) were substituted back into the Gpa1 template and gain-of-function assessed. The C-terminal third of G(16), but not of G(i2), provides sufficient interactions for coupling to occur with PAR1. The N-terminal two-thirds of G(i2) also contains sufficient determinants to bind and couple to PAR1 and overcome the otherwise negative or missing interactions supplied by the C-terminal third of Gpa1. Replacement of the N-terminal alpha-helix of G(i2), residues 1-34, with those of Gpa1 abolishes coupling but not binding to PAR1 or to betagamma subunits. These data support a model that the N-terminal alphaN helix of the Galpha subunit is physically interposed between PAR1 and the Gbeta subunit and directly assists in transferring the signal between agonist-activated receptor and G protein.
Swift
S
S
Molecular Cardiology Research Institute, Division of Hematology, New England Medical Center, Boston, Massachusetts 02111, USA.
Sheridan
P J
PJ
Covic
L
L
Kuliopulos
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D011485:Protein Binding; D011869:Radioligand Assay; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D017931:DNA Primers; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D013917:Thrombin; D019204:GTP-Binding Proteins