Abstract
We have utilized limited in situ trypsinization of the adenylate cyclase-coupled beta-adrenergic receptor of frog erythrocytes to probe the processes of receptor activation, desensitization, and recycling. Treatment of intact erythrocytes with trypsin (1 mg/ml) for 1 h at 20 degrees C converts all the receptor peptides (identified by photoaffinity labeling with p-azido-125I-benzylcarazolol) from a Mr approximately 58,000 to a Mr approximately 40,000 species. Nonetheless, the trypsinized beta-adrenergic receptors bind agonists and antagonists with unaltered affinity and with no change in the number of binding sites. Moreover, the ability of the proteolyzed receptors to interact with the nucleotide regulatory protein to form a high affinity guanine nucleotide-sensitive state and to activate adenylate cyclase were also unaltered. However, upon exposure of intact cells to the agonist isoproterenol, trypsinized beta-adrenergic receptors were more rapidly and more completely cleared from the plasma membranes ("down-regulated") than untrypsinized receptors. Whereas down-regulated receptors from nontrypsinized cells appear to recycle to the cell surface after removal of the agonist, internalized trypsinized beta-adrenergic receptors do not recycle to the plasma membrane and appear to be degraded within the cell. Moreover, when internalized receptors, recovered in a light vesicle fraction, were fused with a heterologous adenylate cyclase system, untreated but not trypsinized receptors reconstituted catecholamine stimulation of the enzyme. These data suggest that the beta-adrenergic receptor contains a trypsin-sensitive site which is exposed on the outer surface of the plasma membrane. Proteolysis at this site releases a fragment which though not critically involved in either ligand binding or "effector coupling" might be important for anchoring the receptors in the plasma membrane. These data also suggest that in situ proteolysis of the receptors might serve as a physiological trigger for their internalization and degradation.
Highlights
We have utilized limited in situ trypsinization of the frog erythrocytes [1,2,3] have indicated that thedesensitization adenylate cyclase-coupled @-adrenergic receptor of of catecholamine-sensitive adenylate cyclase activity is assofrog erythrocytes to probe the processes of receptor ciated with a partial (50%)loss of 0-adrenergic receptor sites activation, desensitization, and recycling
We have previously demonstrated that “down-regulated”@-adrenergic receptors found in thesevesicles are “internalized” in the sense that they are no longer accessible a t the cell surface [3]
When the same experiment was performed with trypsinized erythrocytes, a very different pattern was obtained (Fig. 9B).The @-adrenergicreceptors disappeared from the vesicle fraction (-2 h) as assessed by '251-CYPbinding but failed to reappear in the plasma membrane
Summary
Previous studies utilizing photoaffinity labeling and purification techniques have demonstrated that the @-adrenergic. We have previously been able to quantitativelyrecover the “down-regulated @-adrenergicreceptorsfromfrog erythroprecisemolecular weight estimatessomewhat difficult, the cytes in a “light vesicle” fraction which appears to represent data in Fig. 1 clearly establish that the receptors have been quantitativelyconverted to aform of appreciably smaller internalized orsequestered membranes. As demonstrated inFig. 3, in both untreated and trypsinized membranes the agonist isoproterenol is able to promote the formationof a high affinity nucleotide-sensitive state of the receptor (the “coupled” form of the receptor) as assessed by competition binding with ‘251-CYP.When the guanine nucleotide analog Gpp(NH)pwas present during the binding assay, thcoempetition curves were shifted to the right, indicatiangconversion
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have