A RECEPTOR-G PROTEIN COUPLING-INDEPENDENT STEP IN THE INTERNALIZATIONOF THE THYROTROPIN-RELEASING-HORMONE RECEPTOR

To determine whether functional receptor-G protein coupling or signali ng are required for internalization of the thyrotropin-releasing hormo ne receptor (TRHR), we compared the endocytosis of G(q)-coupled and un coupled receptors. A hemagglutinin epitope-tagged TRHR (HA-TRHR) was i n the G(q)-coupled state when bound to the agonist, MeTRH, and in a no nsignaling state when bound to the HA antibody (12CA5). 12CA5 did not induce an increase in [Ca2+](i) or inositol phosphates and did not inh ibit [H-3]MeTRH binding or MeTRH-induced production of second messenge rs. Both agonist- and antibody-bound HA-TRRRs were rapidly internalize d via the same pathway; internalization was sensitive to hypertonic sh ock, and both types of internalized receptors were sorted into lysosom es. In addition, the amino acid sequence CNC (positions 335-337) in th e C-terminal tail of the TRHR, which is important in ligand-induced re ceptor internalization as determined by deletion mutagenesis (Nussenzv eig, D. R., Heinflink, M., and Gershengorn, M. C. (1993) J. Biol. Chem . 268, 2389-2392), was also important for 12CA5-induced internalizatio n. We expressed two truncated receptors, HA-K338STOP and HA-C335STOP, in GH(1)SC(1) pituitary cells. Both HA-TRHR and HA-K338STOP were local ized at the plasma membrane of untreated cells and were translocated t o intracellular vesicles after MeTRH or 12CA5 binding; however, HA-C33 5STOP was internalized and recycled constitutively. The intracellular localization of HA-C335STOP was not altered by MeTRH; however, 12CA5 b inding induced the disappearance of internalized HA-C335STOP and cause d its localization at the plasma membrane, indicating that constitutiv ely cycling HA-C335STOP cannot be reinternalized after antibody bindin g. Thus, amino acids 335-337, which are important for the internalizat ion of G(q)-coupled TRHRs, are also required for the sequestration of functionally uncoupled TRHRs, and in addition, they act as an inhibito ry signal that prevents constitutive receptor internalization. Specifi cally, the Cys residues at positions 335 and 337 are important for pre venting constitutive TRHR internalization, because a mutant HA-C335S/C 337S receptor was sequestered constitutively. me conclude that release from a negative regulatory internalization sequence or domain is impo rtant for HA-TRHR internalization and that the role of the CNC sequenc e in internalization is independent of functional TRHR-G(q) coupling.