The time-course of agonist-induced solubilization of trimeric G(q)alpha/G(11)alpha proteins resolved by two-dimensional electrophoresis.
Prolonged agonist stimulation results in specific transfer of activated Galpha subunits of G(q)alpha/G(11)alpha family from particulate membrane fraction to soluble (cytosol) cell fraction isolated as 250,000 x g supernatant. In this study, we have used 2D electrophoresis for more defined resolution of Galpha subunits of G(q)alpha/G(11)alpha family and followed the time course of solubilization effect. The small signal of soluble G proteins was already detected in control, hormone-unexposed cells. Hormone stimulation resulted in a slow but continuous increase of both intensity and number of immunoreactive signals/spots of these G proteins (10, 30, 60, 120 and 240 min). At longer times of agonist exposure (>2 hours), a marked increase of G(q)alpha/G(11)alpha proteins was detected. The maximal level of soluble G(q)alpha/G(11)alpha proteins was reached after 16 hours of continuous agonist exposure. At this time interval, eight individual immunoreactive signals of G(q)alpha/G(11)alpha proteins could be resolved. The relative proportion among these spots was 15:42:10:11:7:7:2:5. Solubilization of this class of Galpha proteins was thus observed after prolonged agonist stimulation only, induced by ultra high concentration of hormone and in cells expressing a large number of GPCRs. Our data therefore rather indicate tight/persisting binding of G(q)alpha/G(11)alpha proteins to the membrane.
Durchánková
D
D
Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Novotný
J
J
Svoboda
P
P
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D044385:GTP-Binding Protein alpha Subunits / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D051379:Mice; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000819:agonists / Q000378:metabolism*; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology*; D012995:Solubility; D013997:Time Factors; D014162:Transfection
D044385:GTP-Binding Protein alpha Subunits; D018025:Receptors, Thyrotropin-Releasing Hormone