title: C5a- and ASP-mediated C5L2 activation, endocytosis and recycling are lost in S323I-C5L2 mutation.
abstract: C5L2, a G-protein-coupled receptor (GPCR), has been identified as an ASP (C3adesArg) and C5a receptor. Controversy exists regarding both ligand binding and functionality. ASP activation of C5L2 is proposed to regulate fat storage. C5L2 is also proposed as a decoy receptor for C5a, an inflammatory mediator, based on absence of Ca(2+) or chemotaxis changes.
  
  
  OBJECTIVE
  (i) to evaluate C5L2 receptor activation and recycling using recombinant ASP (rASP) and rC5a and (ii) assess receptor trafficking of S323I-C5L2 mutation previously identified in a family and demonstrated to have altered functionality.
  
  
  RESULTS
  stably transfected C5L2-HEK cells were sorted using fluorescent-ASP (Fluos-ASP) binding. Following 2-h serum-free pretreatment, C5L2 was typically localized to the cell-surface. beta-Arrestin-2-GFP transiently transfected C5L2-HEK cells demonstrated rASP and rC5a-dependent beta-arrestin-2-GFP translocation, which showed time-dependent intracellular colocalization with C5L2. Without ligand or C5L2 transfection, no translocation was identified at any time point. Ligand-dependent (rASP and rC5a) C5L2 endocytosis was time-dependent with a 1-h nadir, and was clathrin- and cholesterol-dependent. Transiently transfected Rab-GFP proteins (Rabs 5, 7 and 11) demonstrated time-dependent colocalization of Rab5, Rab7, and Rab11 with C5L2. In contrast to C5L2, a large proportion of stably transfected S323I-C5L2 did not localize to the cell-surface. While S323I-C5L2 was competent for Fluos-ASP and (125)I-ASP binding, although at a reduced level, there was no ligand-mediated receptor phosphorylation. Further, there was no ligand-mediated activation of beta-arrestin-2-GFP translocation, and no downstream functional activation of glucose transport or triglyceride synthesis.
  
  
  CONCLUSIONS
  C5L2 is a functional metabolic receptor, and serine 323 is important for ASP induced functionality.
authors.affiliation: CRIUCPQ, Université Laval, Québec, Canada.
authors.forename: Wei
authors.initials: W
authors.lastname: Cui
authors.forename: May
authors.initials: M
authors.lastname: Simaan
authors.forename: Stephane
authors.initials: S
authors.lastname: Laporte
authors.forename: Robert
authors.initials: R
authors.lastname: Lodge
authors.forename: Katherine
authors.initials: K
authors.lastname: Cianflone
mesh_terms: D000215:Acylation; D019390:Arrestins / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D015926:Complement C3a / Q000276:immunology* / Q000494:pharmacology; D015936:Complement C5a / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D044087:Receptor, Anaphylatoxin C5a; D019707:Receptors, Chemokine / Q000819:agonists / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000494:pharmacology; D012694:Serine / Q000235:genetics; D014162:Transfection; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D020691:rab GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; C417003:C5aR2 protein, human; D044087:Receptor, Anaphylatoxin C5a; D019707:Receptors, Chemokine; D011994:Recombinant Proteins; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C044541:complement C3a, des-Arg-(77)-; D012694:Serine; D015926:Complement C3a; D015936:Complement C5a; D020691:rab GTP-Binding Proteins