Evidence that constitutively active luteinizing hormone human chorionic gonadotropin receptors are rapidly internalized

The luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, whi ch belongs to the family of G-protein coupled receptors, plays an important role in gonadal steroidogenesis. Substitution of aspartic acid 556 of the LH/hCG receptor with glycine (D556G) creates a constitutively active recept or that activates adenylyl cyclase in the absence of hormone. To examine re ceptor internalization, human embryonic kidney cells (293 T) expressing wil d type (WT) or D556G mutant receptors were incubated with [I-125]hCG and su bsequently analyzed for cell surface bound and internalized radioactivity. Comparison of the rate constants of internalization of the D556G mutant and WT receptors revealed that the rate of internalization of the D556G mutant was five times greater than that of the WT receptor. Although the D556G re ceptor internalizes [I-125]hCG rapidly, a corresponding increase in [I-125] hCG degradation was not seen. The internalization of another constitutively active LH/hCG receptor (aspartic acid 556 to tyrosine) was also greater th an that of the WT receptor. Internalization of receptor bound [I-125]hCG wa s inhibited by a hypertonic sucrose solution, confirming that the ligand en ters the cell by receptor-mediated endocytosis. Furthermore, the constituti vely active D556G and D556Y LH/hCG receptors utilize the arrestin dependent internalization pathway. These results suggest that the active state confo rmation of the constitutively active receptor is conducive to rapid interna lization.