Agonist regulation of β-adrenergic receptors: Immunoblotting and indirect immunofluorescence reveal agonist-induced lateral sequestration and loss of binding

The binding capacity and sub-cellular distribution of β 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 β 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, β ₂ -adrenergic receptors in the plasma membrane fraction were found to decline from ~90% to 50%, while those in a lower-density 'light' vesicle fraction increased from ~10% to 50%. Immunoblotting with specific antisera against the β 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 (β1)-subunit (36,000-Mr) 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. Immunobloting 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 β-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) β-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 β-agonist for a short time (20 min).