Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2.
Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [(125)I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser(449) to Ser(467) were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.
Kuwasako
Kenji
K
Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. kuwasako@fc.miyazaki-u.ac.jp
Kitamura
Kazuo
K
Nagata
Sayaka
S
Hikosaka
Tomomi
T
Kato
Johji
J
D053607:Adrenomedullin / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D058286:Calcitonin Receptor-Like Protein; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C545503:RAMP2 protein, human; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D053607:Adrenomedullin; D000242:Cyclic AMP