Effect of Activating and Inactivating Mutations on the Phosphorylation and Trafficking of the Human Lutropin/Choriogonadotropin Receptor

Abstract

The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact. Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.

GIPC Binds to the Human Lutropin Receptor (hLHR) through an Unusual PDZ Domain Binding Motif, and It Regulates the Sorting of the Internalized Human Choriogonadotropin and the Density of Cell Surface hLHR

“…The preparation and characterization of myc-rLHR-wt-(in pcDNAI/Neo) and myc-hLHR-wt (in pcDNA 3.1)-modified forms of the LHR containing the Myc epitope at the N terminus have also been described (16,17). The different mutants of the rLHR and hLHR used here were constructed by standard PCR strategies using the myc-rLHR-wt or myc-hLHR-wt as templates.…”

“…Down-regulation of the Cell Surface LHR-293T cells transiently transfected with the myc-hLHR-wt or mutants thereof were washed twice with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 1.4 mM KH 2 PO 4 , 4.3 mM Na 2 HPO 4 ), pH 8, and then biotinylated for 30 min at room temperature by incubation with a freshly prepared solution (0.5 mg/ml) of sulfo-NHS-LC-Biotin, (Pierce) as described before (17). After biotinylation, the cells were washed once with Dulbecco's modified Eagle's medium containing 10 mM HEPES and 10% newborn calf serum and incubated for 5 min in the same medium to quench the excess unreacted biotin.…”

“…Some cells were saved on ice and processed immediately (t ϭ 0 samples), whereas others were incubated in 1 ml of warm assay medium containing 52 nM hCG for 6 h at 37°C. At the indicated times, the cells were placed on ice and lysed as described before (17). The receptors were immunoprecipitated with the 9E10 antibody (17).…”

“…At the indicated times, the cells were placed on ice and lysed as described before (17). The receptors were immunoprecipitated with the 9E10 antibody (17). The immunoprecipitates were resolved on SDS gels and electrophoretically transferred to polyvinylidene difluoride membranes (31).…”

A two‐entropies analysis to identify functional positions in the transmembrane region of class A G protein‐coupled receptors

“…Mutation of the positions in cluster 4 can cause receptor constitutive activity, for example, positions 3.43 and 6.37. [37][38][39][40][41][42][43][44] The positions in cluster 5 mostly face the cell membrane. They are divergent both within subfamilies and among all class A GPCRs.…”