Do agonists promote rapid internalization of beta-adrenergic receptors?
We used elution of radioligands at low pH to quantitate intracellular beta-adrenergic receptors on intact S49 lymphoma cells. We validated this method with respect to cell viability, beta-adrenergic receptor integrity, and transferrin receptors on these cells. On control cells, about 15% of the radiolabeled beta-adrenergic antagonists [3H]dihydroalprenolol and [125I]iodocyanopindolol specifically bound at 37 degrees C could not be eluted at low pH; these binding sites appear to be intracellular receptors that are inaccessible to the surface-restricted antagonist [3H]CGP-12177 [tritiated (+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride]. Incubation of cells with the agonist isoproterenol at 37 degrees C for 15 min did not change the number of [3H]dihydroalprenolol binding sites but reduced [3H]CGP-12177 binding sites by 50% or more. However, all specifically bound [3H]CGP-12177 and [3H]dihydroalprenolol were eluted by acid. In addition, the number of acid-elution-resistant [125I]iodocyanopindolol binding sites was not increased in cells coincubated with 1 microM isoproterenol and [125I]iodocyanopindolol for 15 min at 37 degrees C, even though those sites show a loss in apparent affinity for isoproterenol of about 2 orders of magnitude, a loss previously attributed to internalization. We conclude that the early phase of agonist-mediated desensitization of beta-adrenergic receptors in S49 cells does not coincide with the movement of receptors to intracellular sites; instead, agonist-modified receptors remain in association with the plasma membrane and are accessible to the extracellular environment. These "redistributed" receptors together with "cell-surface" and "intracellular" receptors represent three classes of beta-adrenergic receptors that can be selectively identified in intact target cells.
Mahan
L C
LC
Motulsky
H J
HJ
Insel
P A
PA
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D008223:Lymphoma / Q000378:metabolism / Q000648:ultrastructure; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D011990:Receptors, Transferrin
D000318:Adrenergic beta-Agonists; D011943:Receptors, Adrenergic, beta; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin