Decreased Degradation of Internalized Follicle-Stimulating Hormone Caused by Mutation of Aspartic Acid 6.30550 in a Protein Kinase-CK2 Consensus Sequence in the Third Intracellular Loop of Human Follicle-Stimulating Hormone Receptor1

Abstract

A naturally occurring mutation in follicle-stimulating hormone receptor (

Extracellular loop 2 in the FSH receptor is crucial for ligand mediated receptor activation

“…Further, pulse chase experiments were done for determination of internalization, recycling and degradation of labeled hormone in WT FSHR and the EL mutants (Krishnamurthy et al, 2003;Kluetzman et al, 2011). Cells were incubated with medium containing 125 I-FSH.…”

Trafficking of the Follitropin Receptor

“…Add 500 μl of 2 N NaOH to wells at RT and place on orbital shaker for 1 h (at RT) to solubilize cells. Remove and transfer NaOH samples to glass tubes and count in a gamma counter to measure “cell-associated” 125 I-FSH (Kluetzman, Thomas, Nechamen, & Dias, 2011). A protein or DNA assay or cell count on extra wells can be done to normalize binding data with respect to protein concentration or cell number.…”

Normal testicular function without detectable follicle-stimulating hormone. A novel mutation in the follicle-stimulating hormone receptor gene leading to apparent constitutive activity and impaired agonist-induced desensitization and internalization

“…Recycling and degradation of the FSHR were assessed following the procedure described previously (Kluetzman et al, 2011). This assay is a pulse-chase paradigm in which the relative levels of total recycled 125 I-FSH [the sum of trichloroacetic acid (TCA)-insoluble radioactivity in the medium and surface bound radioactivity) and degraded 125 I-FSH (TCA-soluble radioactivity in the medium) are determined during a period of 4 h after allowing internalization of the receptor-ligand complex.…”

“…This assay is a pulse-chase paradigm in which the relative levels of total recycled 125 I-FSH [the sum of trichloroacetic acid (TCA)-insoluble radioactivity in the medium and surface bound radioactivity) and degraded 125 I-FSH (TCA-soluble radioactivity in the medium) are determined during a period of 4 h after allowing internalization of the receptor-ligand complex. At each time point, the recycled 125 I-FSH is an indirect measurement of the amount of internalized receptor that was recycled back to the plasma membrane, whereas the amount of degraded 125 I-FSH represents the fraction of the internalized receptor that was targeted to lysosomes and/or proteasomes for degradation (Kluetzman et al, 2011;Krishnamurthy et al, 2003b).…”

“…Given that agonist-induced early desensitization is followed by internalization of the agonist-bound receptor and that in both processes, b-arrestins play a critical role (Freedman and Lefkowitz, 1996), we next examined the internalization kinetics of the N431I mutant and its regulation by b-arrestin 1 and 2. In equilibrium binding conditions, the internalization of the N431I was significantly delayed as disclosed by the lower disappearance rate of 125 I-FSH [and presumably receptor as well (Kluetzman et al, 2011;Krishnamurthy et al, 2003b)] from the cell surface during a pulse-chase paradigm (slope values of the regression line generated by graphing the surface-associated hormone as a function of time: N431I mutant, À7.76 ± 1.47; WT FSHR, À16.90 ± 3.61; p = 0.02) (Fig. 6A).…”