Abstract
The effects of protein synthesis inhibitors and the lysosomotropic agent chloroquine on the metabolism of the insulin receptor were examined. Through the use of the heavy-isotope density shift technique, cycloheximide was found to inhibit both the synthesis of new insulin receptor and the inactivation of old cellular insulin receptor. Upon investigation of the locus of this effect of protein synthesis inhibition, it was found that cycloheximide did not inhibit 1) the translocation of receptor from the cell surface to an intracellular site, 2) the recycling of receptor from the internal site back to the plasma membrane, nor 3) the degradation of insulin. Cycloheximide did, however, rapidly and completely inhibit the inactivation of the insulin receptor. In the presence of extracellular insulin, this effect of cycloheximide resulted in the long-term (6 h) accumulation of receptor in a trypsin-resistant intracellular compartment. Puromycin and pactamycin, protein synthesis inhibitors with mechanisms of action which differ from cycloheximide, produced the same effects on insulin receptor metabolism as cycloheximide, indicating that this effect on receptor metabolism is due to the inhibition of protein synthesis and not a secondary effect of cycloheximide. Actinomycin D also inhibited the inactivation of receptor. Chloroquine inhibited the receptor-mediated degradation of insulin, but had no effect on either the internalization or inactivation of the insulin receptor. The insulin-induced recycling of the internalized receptor was inhibited by chloroquine, possibly through the inhibition of the discharge of insulin from the insulin-receptor complex. From these observations, we suggest that 1) a protein factor is required to inactivate the insulin receptor, 2) this protein and the messenger RNA coding for the protein have short cellular half-lives, and 3) insulin degradation and insulin receptor inactivation are distinct, separable processes which not only occur at different rates, but possibly occur in distinct subcellular locations.
Highlights
The effects of protein synthesis inhibitors and the ferent rates, butpossibly occur in distinct subcellular lysosomotropicagent chloroquineon the metabolismof locations
Puromycinand pactamycin, protein synthesis inhibitors withmechanisms of action which differ from cycloheximide, produced the same effects on insulin receptor metabolism as cycloheximide, indicating that thiseffect on receptor metabolism is due to the inhibition of protein synthesis andnot a secondassociated with an increased rate constant for receptor inactivation, with no change in the rate of receptor synthesis
The unusual nature of this effect of cycloheximide on receptor down-regulation was noted using the heavy-isotope density shift technique which showed that down-regulation caused increased receptor inactivation with no change in receptor synthetic rates [1,2]
Summary
DIFFERENTIALEFFECTS ON RECEPTOR RECYCLING AND INACTIVATION AND INSULIN DEGRADATION*. From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Surface binding was allowedto equilibrate at least 6 h before washing ers were subjected to the ligand debinding protocol described above, the unbound insulin from the monolayers with 5 volumes of PBS,' but using serum-free cell culture media instead of Krebs-Ringer. The trypsin-containing buffer by polyethylene glycol precipitation [13] All assays included both was aspirated from the dish and replaced with 5 ml of PBS containing total andnonspecific binding and were performed in alteast duplicate 3 mg of soybean trypsin inhibitor. This quench buffer was in each experiment. The mixture was cooled to 4 "C and centrifuged at 7000 X g for 20
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
