Agonist regulation of beta-adrenergic receptors: immunoblotting and indirect immunofluorescence reveal agonist-induced lateral sequestration and loss of binding.

The binding capacity and sub-cellular distribution of beta 2 adrenergic receptors was explored in rat osteosarcoma cells (ROS 17/2.8) following short-term (20 min) and long-term (24 hr) agonist treatment. ROS cells express about 27,000 beta 2 adrenergic receptors per cell, nearly 90% of which appear plasma membrane-associated by radioligand binding analysis. When cells were treated with isoproterenol for 20 min, lysed, and the lysate centrifuged over a gradient of sucrose, beta-adrenergic receptors in the plasma membrane fraction were found to decline from approximately 90% to 50%, while those in a lower-density "light" vesicle fraction increased from approximately 10% to 50%. Immunoblotting with specific antisera against the beta 2 adrenergic receptor revealed, in sharp contrast, that 60% of receptor was distributed in the plasma membrane-enriched fraction, 40% in the light vesicle fraction of untreated cells. A 20-min exposure to agonist caused the 60:40 distribution of immunoreactive receptor between the two fractions (plasma membrane:light vesicle) to shift to 35:65. The distribution of immunoreactive G beta 1-subunit (36,000-M(r)) between the two fractions was 80:20 and not influenced by exposure of cells to agonist. A 24 hr-exposure to agonist changed markedly the distribution of immunoreactive receptor from 60:40 to 90:10 (plasma membrane: light vesicle). Receptor content as determined by radioligand binding, in contrast, was nondetectable in either fraction prepared from ROS cells stimulated by agonist for 24 hr. Immunoblotting of post-nuclear, supernatant fractions of whole-cell lysates revealed no change in receptor content after 24 hr of agonist treatment. Furthermore, SDS-polyacrylamide gel electrophoresis and immunoblotting revealed no prominent proteolytic degradation of receptor in response to agonist stimulation at 20 min or 24 hr. Indirect immunofluorescence of beta adrenergic receptors in fixed, intact ROS cells probed only cell surface-associated receptor and yielded equivalent epifluorescence signals for untreated cells and cells treated with isoproterenol for either 20 min, or 24 hr. The immunological results confirm the phenomenon of agonist-induced receptor sequestration, but reveal several new insights: (i) beta adrenergic receptor protein content and subcellular distribution may not be accurately reflected by radioligand binding; (ii) receptor down-regulation (loss of binding) after 24 hr exposure to agonist cannot be explained by enhanced receptor degradation; (iii) the cell surface complement of receptor is not altered at 20 min or 24 hr following stimulation of cells with agonist; and (iv) lateral sequestration of receptors to separate domains of the cell membrane occurs when ROS 17/2.8 cells are exposed to beta-agonist for a short time (20 min).