Desensitization of CGRP and adrenomedullin receptors in SK-N-MC cells: Implications for the RAMP hypothesis

Recent evidence (1) suggests that the related peptides calcitonin gene-rela ted peptide (CGRP) and adrenomedullin (AM) bind to the same heptahelical tr ansmembrane receptor, with receptor specificity being determined by a recep tor associated modifying protein (RAMP). If correct, this hypothesis would predict that each peptide should desensitize the cellular response to subse quent stimulation by itself or the other peptide. We have therefore studied the patterns of desensitization of these receptors in SK-N-MC cells. SK-N- MC cells were stimulated for 20 minutes in either serum free medium alone ( control) or SFM containing AM 10(-8)M or CGRP 10(-7)M. Cells were then incu bated for a further 20 minutes in SFM containing a second agonist and 1 mM isobutyryl methylxanthine (IBMX), before harvesting and assay for cAMP. Pre -exposure of cells to CGRP or AM decreased cAMP generation in response to s ubsequent stimulation with CGRP by 58% (+/-14) and 42% (+/-14) (SD) respect ively. Pre-incubation of cells with 100 nM H-89 abolished this effect, indi cating that desensitization was mediated through PKA. In contrast, there wa s no attenuation of the cAMP response to stimulation with AM by pre-exposur e to AM or CGRP. These results suggest that CGRP and AM receptors exhibit d ifferent patterns of desensitization in SK-N-MC cells: a finding with signi ficant implications for the RAMP hypothesis.