Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines

Abstract

1. The calcitonin receptor-like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene-related peptide (CGRP) and/or adrenomedullin in transfected cells. 2. There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. 3. We confirmed CGRP subtype 1 receptor (CGRP(1)) pharmacology in SK-N-MC neuroblastoma cells. L6 myoblast cells expressed both CGRP(1) and adrenomedullin receptors whereas Rat-2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP(2) receptor pharmacology for Col-29 colonic epithelial cells, which, instead were CGRP(1)-like in this study. 4. L6, SK-N-MC and Col-29 cells expressed mRNA for RAMP1 and RAMP2 but Rat-2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA. 5. SK-N-MC, Col-29 and Rat-2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus, circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor. 6. These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.

Figure 1

Stimulation of cAMP production in SK-N-MC (a) and Col-29 (b) cells by CGRP in the absence or presence of different CGRP_8-37 concentrations. Cyclic AMP production is shown as pmol cAMP per well. Points represent the mean and s.e.mean of three determinations. (c) Schild plot showing the effects of CGRP_8-37 on SK-N-MC and Col-29 cells. This illustrates the case where the slope of the plot has not been constrained to unity. (d) Effect of [Cys(Et)^2,7]α-CGRP on cAMP production in SK-N-MC and Col-29 cells. Cyclic AMP production is shown as a per cent of the maximal stimulation level in those cells. Points represent the mean and s.e.mean of three determinations.

Figure 2

Northern analysis of Col-29 and SK-N-MC cells showing (from left to right) CRLR, RAMP1, and RAMP2 mRNA. Note that human CRLR mRNA appears as multiple bands, as discussed in the text. The blots were also probed with GAPDH and the resulting autoradiogrphs are aligned under the original blots. The position of the 18S rRNA band, which was used as a marker, is shown on each blot.

Figure 3

Northern analysis of L6-2, L6-1 and Rat-2 cells showing (from left to right) CRLR, RAMP1 and RAMP2 mRNA. These blots were also probed with GAPDH and the resulting autoradiographs are aligned under the original blots. The position of the 18S rRNA band, which was used as a marker, is shown on each blot.

Figure 4

Western blots of rat (a) or human (b) cell line total protein. (a) Twenty μg of total protein analysed on a 10% SDS – PAGE gel was exposed to an anti-rat CRLR antibody. A band of 75 kDa, representing rat CRLR was detected in L6-1, L6-2 and Rat-2 cells. (b) Total protein (20 μg) from human cell lysates was analysed in the same way as for the rat cells. A single band of 80 kDa representing human CRLR was detected in SK-N-MC and Col-29 cells. Molecular weight markers, shown on the left of the blots are: β-galactosidase, 131 kDa; bovine serum albumin, 75 kDa and carbonic anhydrase, 42 kDa.

Figure 5

The effect of passage number on ¹²⁵I-CGRP binding (a), cAMP stimulation (b) and CRLR, RAMP1 and RAMP2 mRNA levels (c) in SK-N-MC cells. (a) Total and non-specific binding (NSB) were measured in the cells as described in Methods. Passage number is shown in parentheses. Each bar represents the mean and s.e.mean of quadruplicate determinations. (b) Cyclic stimulation by CGRP in SK-N-MC cells at passage 5 or 25 expressed as a percentage of the response to 10 μM forskolin (assay points represent the mean of 2 to 3 separate, duplicate experiments, vertical lines are s.e.mean). The cAMP production in response to forskolin was 30 pmol per well (passage 5) and 6.5 pmol per well (passage 25). (c) Northern analysis of low passage (5) or high passage (25) SK-N-MC cells showing (from left to right) CRLR, RAMP1 and RAMP2 mRNA. These blots were also probed with GAPDH and the resulting autoradiographs are aligned under the original blots. The position of the 18S rRNA band, which was used as a marker, is shown on each blot.