title: The third extracellular loop of the human calcitonin receptor-like receptor is crucial for the activation of adrenomedullin signalling.
abstract: OBJECTIVE
  The extracellular loops (ECLs) in Family A GPCRs are important for ligand binding and receptor activation, but little is known about the function of Family B GPCR ECLs, especially ECL3. Calcitonin receptor-like receptor (CLR), a Family B GPCR, functions as a calcitonin gene-related peptide (CGRP) and an adrenomedullin (AM) receptor in association with three receptor activity-modifying proteins (RAMPs). Here, we examined the function of the ECL3 of human CLR within the CGRP and AM receptors.
  
  
  METHODS
  A CLR ECL3 chimera, in which the ECL3 of CLR was substituted with that of VPAC2 (a Family B GPCR that is unable to interact with RAMPs), and CLR ECL3 point mutants were constructed and transiently transfected into HEK-293 cells along with each RAMP. Cell-surface expression of each receptor complex was then measured by flow cytometry; [(125) I]-CGRP and [(125) I]-AM binding and intracellular cAMP accumulation were also measured.
  
  
  RESULTS
  Co-expression of the CLR ECL3 chimera with RAMP2 or RAMP3 led to significant reductions in the induction of cAMP signalling by AM, but CGRP signalling was barely affected, despite normal cell-surface expression of the receptors and normal [(125) I]-AM binding. The chimera had significantly decreased AM, but not CGRP, responses in the presence of RAMP1. Not all CLR ECL3 mutants supported these findings.
  
  
  CONCLUSIONS
  The human CLR ECL3 is crucial for AM-induced cAMP responses via three CLR/RAMP heterodimers, and activation of these heterodimers probably relies on AM-induced conformational changes. This study provides a clue to the molecular basis of the activation of RAMP-based Family B GPCRs.
authors.affiliation: Frontier Science Research Center, University of Miyazaki, Miyazaki, Japan. kuwasako@fc.miyazaki-u.ac.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Kuwasako
authors.forename: Debbie L
authors.initials: DL
authors.lastname: Hay
authors.forename: Sayaka
authors.initials: S
authors.lastname: Nagata
authors.forename: Tomomi
authors.initials: T
authors.lastname: Hikosaka
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kitamura
authors.forename: Johji
authors.initials: J
authors.lastname: Kato
mesh_terms: D053607:Adrenomedullin / Q000378:metabolism*; D058286:Calcitonin Receptor-Like Protein / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D017354:Point Mutation; D011485:Protein Binding; D058263:Receptor Activity-Modifying Protein 2 / Q000378:metabolism*; D058264:Receptor Activity-Modifying Protein 3 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D058286:Calcitonin Receptor-Like Protein; D008024:Ligands; C545503:RAMP2 protein, human; C545504:RAMP3 protein, human; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D053607:Adrenomedullin; D000242:Cyclic AMP