THE CYTOPLASMIC DOMAIN OF C-CAM IS REQUIRED FOR C-CAM-MEDIATED ADHESION FUNCTION - STUDIES OF A C-CAM TRANSCRIPT CONTAINING AN UNSPLICED INTRON

Cell-CAM]05 (also named C-CAM) is a cell surface glycoprotein involved in intercellular adhesion of rat hepatocytes. It has four extracellul ar immunoglobulin (Ig) domains, a transmembrane domain and a cytoplasm ic domain and therefore is a member of the Ig supergene family. We hav e characterized multiple cDNAs of the C-CAM genes in rat intestine. Se quence analyses showed that rat intestine contained not only the previ ously reported L-form and S-form C-CAMs (renamed C-CAM1 and C-CAM2 res pectively) but also a new isoform, C-CAM3. The C-CAM3 transcript codes for a polypeptide with a truncated C-terminus that lacks 65 amino aci ds from the previously reported C-CAM1 cytoplasmic domain. Unlike C-CA M1, C-CAM3 did not mediate cell adhesion when expressed in insect cell s using the baculoviral expression system. Thus the extra 65 amino aci ds in the cytoplasmic domain of C-CAM1 are important for adhesion phen otype when expressed in insect cells. Although C-CAM1 and C-CAM2 are e ncoded by different genes, sequence analysis suggests that C-CAM3 is p robably derived from alternative splicing of the C-CAM1 gene. To exami ne this possibility, we have determined the exon organization of the C -CAM1 gene. C-CAM3 differed from C-CAM1 by the presence of a single un spliced intron which contained a stop codon immediately after the regu lar splice junction. As a result, translation of C-CAM3 terminates at the point where C-CAM1 and C-CAM3 sequences diverge. To investigate th e expression of C-CAM1, C-CAM2 and C-CAM3 in different tissues, we use d an RNAse-protection assay to simultaneously assess the levels of exp ression of these transcripts. Using total RNA prepared from various ti ssues, we showed that expression of C-CAM3 was tissue-specific, and th e C-CAM3 transcript accounted for about 25% of the transcripts derived from the C-CAM1 gene. However, further analysis revealed that C-CAM3 transcript was not present in cytosolic RNA, rather it was enriched in nuclear RNA prepared from hepatocytes. Although C-CAM3 cDNA contains the polyadenylation signal and is polyadenylated, these results indica te that C-CAM3 is probably an incomplete spliced product of C-CAM1 gen e.