Calcitonin gene-related peptide (CORP) and adrenomedullin (AM) are potent vasodilators that activate the calcitonin-like receptor complexed with one of three receptor activity modifying proteins (RAMP1 for CORP, RAMP 2/3 for AM). Desensitisation of these receptors was characterised in the human neuroblastoma SK-NMC cell line. Desensitisation was dose-dependent (ICso values: CORP, 2.92 x 10-7 M; AM, 1.08 x 10-7 M) and time-dependent, being maximal with a 2-hour agonist pretreatment. Inhibitor experiments showed that CORP-induced desensitisation was independent of protein kinase A (H-89) but was partially mediated by protein kinase C (OFI09203X). Lipid raft-mediated receptor endocytosis did not seem to contribute to signal attenuation (methyl-f3-cyclodextrin and filipin), but clathrin-mediated internalisation plays a role (sucrose). Stable overexpression of dominant-negative ORK2 or dynamin had no effect on desensitisation, suggesting that ORKs and dynamin are not involved. Unlabelled CORP competed 2-[12sI]iodohistidyllO-hCORP (ICso=1.97 x 10-10 M). 15% 12SI_CORP internalised after 10 min, 47% after 120 min. Receptor endocytosis was not blocked by lipid raft inhibitors but was significantly reduced by sucrose, in line with the hypothesis that its effect on desensitisation was due to inhibition of clathrin-mediated internalisation. Overexpression of dominant-negative ORK2 or Dyn 1 did not alter internalisation, suggesting that ORKs and dynamin are not involved. CORP receptor endocytosis was confirmed by confocal immunofluorescence microscopy and biotin internalisation. f3-arrestin 2-eYFP did not redistribute following CORP stimulation. Cells stably expressing short hairpin RNA against f3-arrestinl and/or 2 were generated. Maximum silencing of f3-arrestinl mRNA was 29.8%. 48.2% f3-arrestin2 mRNA knockdown was achieved, with possible reduction of protein. CORP receptor desensitisation was unchanged compared to wild-type cells, consistent with the hypothesis that f3-arrestin2 is not required. Taken together, the data demonstrate desensitisation of the SK-N-MC CORP and AM receptors. CORP receptor desensitises through a PKC-mediated mechanism involving internalisation via clathrin-coated vesicles.