Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

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• 作者：Kenji Kuwasako ; Kazuo Kitamura ; Sayaka Nagata ; Tomomi Hikosaka ; Johji Kato
• 关键词：Adrenomedullin ; Calcitonin receptor-like receptor ; Receptor activity-modifying protein 2 ; Receptor mutations ; Signal transduction ; Receptor internalization
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2010
• 出版时间：12 February 2010
• 年：2010
• 卷：392
• 期：3
• 页码：380-385
• 全文大小：426 K

文摘

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 [¹²⁵I]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 (100 ng/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 Ser⁴⁴⁹ to Ser⁴⁶⁷ 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.