Receptor and membrane interaction sites on Gbeta. A receptor-derived peptide binds to the carboxyl terminus.
The functional organization of Gbetagamma is poorly understood. Regions of bovine brain Gbetagamma that interact with a photoaffinity derivative of an alpha2-adrenergic receptor-derived peptide from the third intracellular loop (diazopyruvoyl-modified peptide Q (DAP-Q)) and a hydrophobic membrane probe (3-trifluoromethyl-3-(m-iodophenyl)diazirine (TID)) were examined. We previously showed that DAP-Q cross-links to specific, competable sites on both the alpha and beta subunits of Go/Gi but not on the gamma subunit and that betagamma subunit was required for stimulation of Go/Gi GTPase activity (Taylor, J. M., Jacob Mosier, G. G., Lawton, R. G., Remmers, A. E., and Neubig, R. R. (1994) J. Biol. Chem. 269, 27618-27624). Similarly, we show here that the membrane-associated photoprobe [125I]TID labels alpha and beta but not gamma. We have now mapped the sites of incorporation of DAP-Q and TID into the beta subunit. TID labels both the 14-kDa amino-terminal and the 23-kDa carboxyl-terminal fragments from a partial tryptic digest of beta while DAP-Q labels only the carboxyl-terminal fragment. Further mapping with endopeptidase Lys C reveals substantial labeling of multiple fragments by TID while DAP-Q labels predominantly a approximately 6-kDa fragment within the carboxyl-terminal 60 amino acids of beta1. Thus, regions within the 7th (or possibly 6th) WD-40 repeat of the beta subunit of G protein interact with the receptor-derived peptide while membrane interaction involves multiple sites throughout the beta subunit.
Taylor
J M
JM
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Jacob-Mosier
G G
GG
Lawton
R G
RG
VanDort
M
M
Neubig
R R
RR
D000818:Animals; D001389:Azirines; D001391:Azo Compounds; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010782:Photolysis; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin
D001389:Azirines; D001391:Azo Compounds; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D018341:Receptors, Adrenergic, alpha-2; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D014357:Trypsin; D019204:GTP-Binding Proteins