Previous work indicated that GTP-binding proteins (G-proteins) are associated as multimeric proteins with the plasma membrane of cells. The theory was proposed that coupling of receptors to multimeric G- proteins can be likened to the association of myosin with multimeric actin; regulation of the latter is governed by the binding and hydrolysis of ATP to ADP whereas receptor-G protein coupling is governed by the concerted binding of hormone and GTP, and degradation of the latter to DGP. In other theories of hormonal activation of G proteins, the products of activation are the dissociated alpha and betagamma subunits of the heterotrimeric G proteins. Using digitonin for extracting multimeric G proteins, we have found that the products of activation of various species of G proteins extracted from brain membranes are monomoers (i.e., heterotrimers) rather than dissociated alpha and betagamma subunits as observed when the same membranes are extracted with Lubrol, which is generally used for extraction. These findings raise new issues regarding the nature of the activation of various effector systems by G proteins. In a second line of investigation, polyclonal antibodies raised against several types of G proteins were used for determining the cellular disposition of G proteins utilizing both normal optics and confocal microscopy for immunodetection of the alpha-subunits of the G proteins. One type, Galphai, was found in three different cell types to be located in the cytosolic compartment in the form of punctate structures, possibly vesicular in nature. In 3T3 fibroblasts, the punctate structures are associated with stress fibers indicating an association with the cytoskeletal matrix; microtubulin filaments were not prominently associated with Galphai. These studies raise fundamental questions regarding how G proteins function in cells not only as transducers linked to surface receptors but possibly also as regulators of movement and targeting of vesicles associated with the cytoskeletal matrix of cells.
| Putney Jr, J W (1999) ""Kissin' cousins"": intimate plasma membrane-ER interactions underlie capacitative calcium entry. Cell 99:5-8 |
