Restricting the mobility of Gs alpha: impact on receptor and effector coupling.
The alpha-subunit of the stimulatory G protein, Gs, has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of Gs alpha dissociation, we constructed a membrane-tethered Gs alpha (tetGs alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gs alpha, tetGs alpha coupled much more efficiently to the beta 2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when Gs )alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGS alpha to adenylyl cyclase. These results demonstrate that regulating the mobility of Gs alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetGS alpha can facilitate the study for receptor-G protein interactions.
Lee
T W
TW
Howard Hughes Medical Institute, Division of Cardiovascular Medicine, Stanford University Medical School, California 94305-5345, USA.
Seifert
R
R
Guan
X
X
Kobilka
B K
BK
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism; D058666:Adrenergic beta-2 Receptor Agonists; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000235:genetics; D011956:Receptors, Cell Surface / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017447:Receptors, Dopamine D1 / Q000819:agonists / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology
D058666:Adrenergic beta-2 Receptor Agonists; D018343:Receptors, Adrenergic, beta-2; D011956:Receptors, Cell Surface; D017447:Receptors, Dopamine D1; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D007545:Isoproterenol