Photoaffinity Identification of Colchicine‐Solubilized Regulatory Subunit from Rat Brain Adenylate Cyclase

Abstract

Five GTP binding proteins in rat cerebral cortex synaptic membranes were identified by photoaffinity labelling with [3H] or [32P](P3-azido-anilido)-P1-5' GTP (AAGTP). When AAGTP-treated membranes were incubated with colchicine or vinblastine and subsequently washed, a single AAGTP-labelled protein of 42 kD was released into the supernatant. About 30% of the total labelled 42-kD protein was released into supernatants from membranes pretreated with colchicine or vinblastine compared with 15% released from control membranes. The amount of adenylate cyclase regulatory subunit (G unit) remaining in these membranes was assessed with reconstitution studies after inactivating the adenylate cyclase catalytic moiety with N-ethylmaleimide (NEM). Forty to fifty percent of functional G units were lost from membranes treated with colchicine prior to washing. This 40-50% loss of functional G unit after colchicine treatment corresponds to the previously observed 42% loss of NaF and guanylyl-5'-imidodiphosphate [Gpp(NH)p]-activated adenylate cyclase. Release of the AAGTP-labelled 42-kD protein from colchicine-treated synaptic membranes is double that from lumicolchicine-treated membranes. This colchicine-mediated release of 42-kD protein correlates with a doubling of functional G unit released from synaptic membranes after colchicine treatment. These findings suggest multiple populations of the G unit within the synaptic plasma membrane, some of which may interact with cytoskeletal components.

Chronic Treatment of C6 Glioma Cells with Antidepressant Drugs Results in a Redistribution of Gsα

“…6B). Previous studies have suggested that activated Gs␣ can be released from the plasma membrane into the cytosol; these results are certainly consistent with those observations (Rasenick et al, 1984;Ransas et al, 1989;Levis and Bourne, 1992). Furthermore, the distribution of Go␣ was not modified by antidepressant treatment (Fig.…”

Real-Time Visualization of a Fluorescent Gαs: Dissociation of the Activated G Protein from Plasma Membrane

“…Previous studies, using cell fractionation or observations of fixed cells, have suggested that activated G␣ s may be released from plasma membranes (Rasenick et al, 1984;Ransas et al, 1989;Levis and Bourne, 1992). In this study, cholera toxin-activated G␣ s -GFP was released from plasma membrane during the course of activation and were gradually distributed through the cytosol.…”

“…Previous studies, using cell fractionation and immunocytochemical assays, suggested that activation by ␤ adrenergic agonist, cholera toxin or direct binding of a hydrolysis-resistant GTP analog resulted in G␣ s translocation from plasma membrane to cytoplasm (Rasenick et al, 1984;Ransas et al, 1989;Levis and Bourne, 1992). G␣ q/11 has also been reported to transiently translocate to plasma membrane of adrenal glomerulosa cells in response to stimulation by angiotensin II for 1 to 5 min (Cote et al, 1997).…”

Chronic Antidepressant Treatment Prevents Accumulation of Gsα in Cholesterol-Rich, Cytoskeletal-Associated, Plasma Membrane Domains (Lipid Rafts)

“…It is well established that treatment of cells with microtubule-disrupting agents like colchicine and vinblastine leads to an increase in G protein-mediated activation of adenylyl cyclase (Kennedy and Insel, 1979;Rasenick et al, 1981Rasenick et al, , 1984Leiber et al, 1993;Jasper et al, 1995;Nishigaki et al, 1998;Rudolph et al, 1979). These potential actions would allow the cAMP signal to be propagated after receptor desensitization mediated by antidepressant treatment (reviewed by Sulser, 1984).…”