Immature human osteoblastic MG63 cells predominantly express a subtype 1-like CGRP receptor that inactivates extracellular signal response kinase by a cAMP-dependent mechanism.

Although accumulated data suggest that calcitonin gene-related peptide (CGRP) produces anabolic effects in skeletal tissue by directly acting on osteogenic cells, neither the distribution of CGRP receptor subtypes nor the associated cellular signaling pathways are well understood. In this study, we have pharmacologically and biochemically characterized CGRP-binding sites in immature human osteoblastic MG63 cells. In a [125I]CGRP whole-cell-binding assay, nonlinear regression curve-fitting analysis demonstrated a single binding site (K(D)=405+/-29 pM; 13,100+/-223 sites per cell). Immunocytochemical and Western blot analyses demonstrated that 48-, 52-, and 120-kDa forms of the calcitonin receptor-like receptor (CRLR) and a 15-kDa form of the receptor-activity-modifying protein-1 (RAMP-1) was expressed on the plasma membrane. CGRP strongly stimulated cellular cAMP production and this effect was antagonized not only by an antagonist of the subtype-1 CGRP (CGRP(1)) receptor, CGRP-(8-37), but by an agonist of the putative subtype-2 CGRP (CGRP(2)) receptor, [Cys(Acm)(2,7)]-CGRP, that also itself acted as a weak agonist. In contrast to published data, CGRP dose- and time-dependently dephosphorylated and inactivated extracellular signal response kinase (ERK). This action was blocked by CGRP-(8-37), by an inhibitor of cAMP-dependent protein kinase (H-89), or by an inhibitor of protein phosphatases (vanadate). Prolonged CGRP treatments significantly suppressed DNA synthesis at 27 h, but up-regulated type I collagen. Both these actions were blocked by CGRP-(8-37) and mimicked by a specific inhibitor of ERK (PD98059). In summary, our data suggest that the CGRP receptors in MG63 cells meet many, but not all, of the classical criteria used to define CGRP(1) receptors. These receptors that functioned in a pharmacologically distinct manner could inhibit cell proliferation, and were substantially more sensitive to a CGRP(2) receptor agonist than are typical CGRP(1) receptors. These receptor proteins were not exactly matched with the known components of a CGRP(1) receptor that have been reported. Therefore, it is possible that the CGRP receptors expressed in immature osteoblastic human MG63 cells represent a variation of the known CGRP(1) receptor.