Direct insertion and fluorescence studies of rhodamine-labeled beta-adrenergic receptors in cell membranes.
Previous studies utilizing the fluorescence of propanolol as a probe for the beta-adrenergic receptor showed that this receptor is motionally constrained. To further study the beta-adrenergic receptor in situ we have reinserted rhodamine-labeled beta-receptors into cell membranes. This report presents documentation of their insertion and physiologic viability. Beta-receptors were purified by affinity chromatography (10,000-fold), then fluorescently labeled with tetramethyl rhodamine isothiocyanate, repurified (55,000-fold) and incubated with rat pancreatic islet cells. The binding of 3H-dihydroalprenolol by these cells was increased from a Bmax of 168 +/- 2 to 309 +/- 20 fmol/mg protein with no change in Kd. Various treatments which remove peripheral membrane proteins, e.g. NaOH, lithium diiodosalicylate, and trypsinization, did not alter binding by the cells with inserted receptors indicating that the receptors inserted into cell membranes. In islet cells treated with Koshland's reagent I, beta-adrenergic binding was completely abolished, but following incubation with isolated beta-receptors, the cells bound beta-adrenergic radioligand with a Bmax of 100 fmol/mg protein, indicating functionality on the part of the inserted receptors. Furthermore, insertion of isolated receptors into frog erythrocytes resulted in increased production of cAMP in response to added isoproterenol. In pancreatic islet cells, incubation with labeled receptors caused the fluorescence to shift in wavelength with increased intensity indicating a shift from an aqueous to a lipid environment, probably into the membrane. Using fluorescence (P), it was found that the inserted receptors became motionally constrained to a P of 0.38 (limiting Po = 0.42) during the first 15 min, remaining so for at least 2 hr. Colchicine (5 micrograms/ml) caused a decrease in P to 0.18 indicating release of constraint. Isoproterenol (10(-5)M) caused a rapid decrease to P = 0.15. This effect was blocked by propranolol. Propranolol itself (10(-5) M) had no effect. These results indicate that the labeled receptors rapidly insert into cell membranes and also support the view that agonist activation of the receptor causes an increase in receptor mobility, presumably due to release of constraint from cytoskeleton elements.
Cherksey
B D
BD
Mendelsohn
S A
SA
Zadunaisky
J A
JA
Altszuler
N
N
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000097:blood; D004082:Dihydroalprenolol / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism* / Q000648:ultrastructure; D007515:Islets of Langerhans / Q000378:metabolism*; D007545:Isoproterenol / Q000378:metabolism; D007700:Kinetics; D008297:Male; D011892:Rana catesbeiana; D051381:Rats; D011919:Rats, Inbred Strains; D011943:Receptors, Adrenergic, beta / Q000302:isolation & purification / Q000378:metabolism*; D012235:Rhodamines; D014316:Tritium
D011943:Receptors, Adrenergic, beta; D012235:Rhodamines; D014316:Tritium; D004082:Dihydroalprenolol; D000242:Cyclic AMP; D007545:Isoproterenol