Functions of the cytoplasmic tails of the human receptor activity-modifying protein components of calcitonin gene-related peptide and adrenomedullin receptors.
Receptor activity-modifying proteins (RAMPs) enable calcitonin receptor-like receptor (CRLR) to function as a calcitonin gene-related peptide receptor (CRLR/RAMP1) or an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Here we investigated the functions of the cytoplasmic C-terminal tails (C-tails) of human RAMP1, -2, and -3 (hRAMP1, -2, and -3) by cotransfecting their C-terminal deletion or progressive truncation mutants into HEK-293 cells stably expressing hCRLR. Deletion of the C-tail from hRAMP1 had little effect on the surface expression, function, or intracellular trafficking of the mutant heterodimers. By contrast, deletion of the C-tail from hRAMP2 disrupted transport of hCRLR to the cell surface, resulting in significant reductions in (125)I-hAM binding and evoked cAMP accumulation. The transfection efficiency for the hRAMP2 mutant was comparable with that for wild-type hRAMP2; moreover, immunocytochemical analysis showed that the mutant hRAMP2 remained within the endoplasmic reticulum. FACS analysis revealed that deleting the C-tail from hRAMP3 markedly enhances AM-evoked internalization of the mutant heterodimers, although there was no change in agonist affinity. Truncating the C-tails by removing the six C-terminal amino acids of hRAMP2 and -3 or exchanging their C-tails with one another had no effect on surface expression, agonist affinity, or internalization of hCRLR, which suggests that the highly conserved Ser-Lys sequence within hRAMP C-tails is involved in cellular trafficking of the two AM receptors. Notably, deleting the respective C-tails from hRAMPs had no effect on lysosomal sorting of hCRLR. Thus, the respective C-tails of hRAMP2 and -3 differentially affect hCRLR surface delivery and internalization.
Kuwasako
Kenji
K
First and Third Departments of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Miyazaki 889-1692, Japan. kuwasako@fc.miyazaki-med.ac.jp
Cao
Yuan-Ning
YN
Chu
Chun-Ping
CP
Iwatsubo
Shuji
S
Eto
Tanenao
T
Kitamura
Kazuo
K
D000595:Amino Acid Sequence; D015153:Blotting, Western; D015740:Calcitonin Gene-Related Peptide / Q000737:chemistry*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation; D000242:Cyclic AMP / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018000:Receptors, Peptide / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C545502:RAMP1 protein, human; C545503:RAMP2 protein, human; D012333:RNA, Messenger; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; D004247:DNA; D003513:Cycloheximide; D000242:Cyclic AMP; D015740:Calcitonin Gene-Related Peptide; D008239:Lysine