Desensitization of CGRP and Adrenomedullin Receptors in SK-N-MC Cells: Implications for the RAMP Hypothesis

Abstract

Recent evidence (1) suggests that the related peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) bind to the same heptahelical transmembrane receptor, with receptor specificity being determined by a receptor associated modifying protein (RAMP). If correct, this hypothesis would predict that each peptide should desensitize the cellular response to subsequent 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 incubated 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 subsequent stimulation with CGRP by 58% (+/-14) and 42% (+/-14) (SD) respectively. Pre-incubation of cells with 100 nM H-89 abolished this effect, indicating that desensitization was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with AM by pre-exposure to AM or CGRP. These results suggest that CGRP and AM receptors exhibit different patterns of desensitization in SK-N-MC cells: a finding with significant implications for the RAMP hypothesis.

Adrenomedullin in the rostral ventrolateral medulla inhibits baroreflex control of heart rate: a role for protein kinase A

“…As the cAMP‐PKA pathway mediates some functions of ADM in vitro (Drake et al ., 1999; Spampinato et al ., 1999; Xu & Krukoff, 2005), we investigated whether the PKA‐related signaling is required for ADM's effect on baroreflex activity. We show that the inhibition of the baroreflex induced by 1 fmol ADM was completely eliminated by coinjections of 100 pmol H‐89, a PKA inhibitor, whereas H‐89 alone had no significant effect on the baroreflex (Figure 5 and Table 1).…”

“…Our data show that coinjections of H‐89 with ADM abolished the ADM‐induced inhibition of the baroreflex to implicate PKA as a mediator of ADM's effect on the baroreflex, although we cannot rule out the possibility that a small part of the effect of H‐89 is due to nonspecific effects on other intracellular messengers such as protein kinase G (Satake et al ., 1996) and Rho kinase (Leemhuis et al ., 2002). Consistent with our results, it has been shown that ADM elevates intracellular cAMP levels in various types of cells (Kitamura et al ., 1993; Shimekake et al ., 1995), including neural cells (Drake et al ., 1999; Spampinato et al ., 1999; Xu & Krukoff, 2005), and that many of ADM's actions are mediated by the cAMP‐PKA pathway in vitro (Drake et al ., 1999; Spampinato et al ., 1999; Xu & Krukoff, 2005).…”

“…Thus, baroreflex curves were generated using a pharmacological pressor agent (phenylephrine, PHEN) and a depressor agent (nitroprusside, NP) before and after ADM and/or its antagonist were microinjected into the RVLM of anesthetized rats. Furthermore, since the cyclic adenosine monophosphate (cAMP)‐protein kinase A (PKA) pathway has been shown to mediate ADM's actions in vitro (Drake et al ., 1999; Spampinato et al ., 1999; Xu & Krukoff, 2005), we coinjected a PKA inhibitor (H‐89) with ADM to determine whether PKA is required for ADM's effects on baroreflex activity.…”

Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays

“…It is preferable to develop assays that use cells endogenously expressing receptor and measure an intrinsic response to best mimic an in vivo setting. CGRP can signal through adenylate cyclase to increase cAMP levels, 5 and we chose to use a well-established CGRP cAMP assay with SK-N-MC cells, 30 which endogenously express CALRLR and RAMP1, for functional profiling of purified scFv and IgG. This provided a robust high-throughput (384-well) format for profiling large numbers of samples in a cell-based assay.…”

Immunomodulatory Role of Neuropeptides in the Cornea

“…The desensitization of the signals and cAMP responses are attenuated after a second exposure to CGRP due to the activity of PKA and PKC [ 316 , 317 , 318 ]. After the transient stimulation of the receptor, the CLR is phosphorylated and the β-arrestin complex is internalized to clathrin-coated pits for endocytosis and rapidly recycled back to the plasma membrane [ 319 ].…”