Two receptor activity-modifying proteins (RAMP2 and RAMP3) enable calcitonin receptor-like receptor (CLR) to function as two heterodimeric receptors (CLR/RAMP2 and CLR/RAMP3) for adrenomedullin (AM), a potent cardiovascular protective peptide. Following AM stimulation, both receptors undergo rapid internalization through a clathrin-dependent pathway, after which CLR/RAMP3, but not CLR/RAMP2, can be recycled to the cell surface for resensitization. However, human (h)RAMP3 mediates CLR internalization much less efficiently than does hRAMP2. Therefore, the molecular basis of the single transmembrane domain (TMD) and the intracellular domain of hRAMP3 during AM receptor internalization was investigated by transiently transfecting various RAMP chimeras and mutants into HEK-293 cells stably expressing hCLR. Flow cytometric analysis revealed that substituting the RAMP3 TMD with that of RAMP2 markedly enhanced AM-induced internalization of CLR. However, this replacement did not enhance the cell surface expression of CLR, [I-125]AM binding affinity or AM-induced cAMP response. More detailed analyses showed that substituting the Thr(130)-Val(131) sequence in the RAMP3 TMD with the corresponding sequence (Ile(157)-Pro(158)) from RAMP2 significantly enhanced AM-mediated CLR internalization. In contrast, substituting the RAMP3 target sequence with Ala(130)-Ala(131) did not significantly affect CLR internalization. Thus, the RAMP3 TMD participates in the negative regulation of CLR/RAMP3 internalization, and the aforementioned introduction of the Ile-Pro sequence into the RAMP3 TMD may be a strategy for promoting receptor internalization/resensitization. (C) 2012 Elsevier Inc. All rights reserved.