Characterization of the single transmembrane domain of human receptor activity-modifying protein 3 in adrenomedullin receptor internalization

Journal Article · Fri Apr 13 04:00:00 EDT 2012 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2012.03.037· OSTI ID:22207803

Kuwasako, Kenji ^[1]; Kitamura, Kazuo; Nagata, Sayaka ^[2]; Nozaki, Naomi; Kato, Johji ^[1]

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1. Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, Miyazaki 889-1692 (Japan)
2. Division of Circulation and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, Miyazaki 889-1692 (Japan)

Highlights: Black-Right-Pointing-Pointer RAMP3 mediates CLR internalization much less effectively than does RAMP2. Black-Right-Pointing-Pointer The RAMP3 TMD participates in the negative regulation of CLR/RAMP3 internalization. Black-Right-Pointing-Pointer A new strategy of promoting internalization and resensitization of the receptor was found. -- Abstract: 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, [{sup 125}I]AM binding affinity or AM-induced cAMP response. More detailed analyses showed that substituting the Thr{sup 130}-Val{sup 131} sequence in the RAMP3 TMD with the corresponding sequence (Ile{sup 157}-Pro{sup 158}) from RAMP2 significantly enhanced AM-mediated CLR internalization. In contrast, substituting the RAMP3 target sequence with Ala{sup 130}-Ala{sup 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.

• OSTI ID: 22207803
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 3 Vol. 420; ISSN 0006-291X; ISSN BBRCA9
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
AMP
CALCITONIN
CELL PROLIFERATION
CHIMERAS
FLUORESCENCE
GENE REGULATION
IODINE 125
RECEPTORS
STIMULATION