MOLECULAR CHARACTERIZATION OF CALCITONIN-GENE-RELATED PEPTIDE (CGRP) IN A RAT MEDULLARY-THYROID CARCINOMA CELL-LINE

The rat medullary thyroid carcinoma cell line, CA-77, is known to expr ess the calcitonin gene and the cell line has been used for characteri zation of procalcitonin. The present investigations concentrate on a m olecular characterization of the calcitonin gene-related peptide (CGRP ) expressed by a subclone of this cell line. The investigations demons trate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogen eity for both CGRP and calcitonin, but a significant amount of immunor eactivity elutes corresponding to the elution position for synthetic C GRP and calcitonin, respectively. The gel chromatogram for CGRP demons trates four immunoreactive peaks with K-d of 0.42, 0.53, 0.68, and 0.8 5. The immunoreactive peak with K-d 0.42 elutes corresponding to synth etic rat CGRP. The four immunoreactive peaks were characterized by hig h pressure liquid chromatography followed by sequence analysis and mas s spectrometry, The immunoreactive peak with K-d 0.42 was identified a s rat alpha-CGRP as was the peak with K-d 0.53. The peak with K-d 0.68 was identified as 19-37 rat alpha-CGRP and the peak with K-d 0.85 as 28-37 rat alpha-CGRP. In summary, we find that the CA-77 cell line exp resses large quantities of normally processed amidated alpha-CGRP and specific fragments thereof. However, the cell line does not express de tectable levels of rat beta-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expressio n.