We report that compartmentalisation of the stimulatory guanine‐nucleotide‐binding regulatory protein (Gs) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the α subunit of Gs (Gsα) [Ransnäs, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900–7903], three membrane‐bound forms of Gsα were identified through ratezonal centrifugation in sucrose density gradients, Gsα‐specific anti‐peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of Gsα in the high‐density portion of the gradient (1.13–1.16 g/cm3) is identical with that of β‐adrenergic‐receptor binding, Na/K‐ATPase and adenylate cyclase activity, and may therefore be identified as plasma‐membrane fragments. The second pool, which was recovered in the middle portion of the gradient (1.09–1.11 g/cm3), contains a much lower total amount of Gsα and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH–cytochrome‐c reductase and glucose‐6‐phosphatase.
The identity of the third pool of Gsα located at the top of the gradient (1.06–1.08 g/cm3), is unknown. The Golgi apparatus marker, UDPgalactose:N‐acetylglucosamine glycosyltransferase, was partially recovered in this area; however, this enzyme was also present in the high‐density portion of the gradient. Complete absence of specific adenylate cyclase and Na/K‐ATPase activity indicates that this low‐density (light) membrane form of Gsα is distinct from any plasma‐membrane fragments. Furthermore, sedimentation at 100000 ×g proves its particulate (membrane) character. The light membrane form of Gsα subunit is functionally active in an adenylate cyclase complementation assay using cyc− membranes devoid of Gsα.
Overall, our data indicates that a substantial portion of Gsα is localized in membrane pools other than plasma membrane.