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-CAM105 (also named C-CAM) is a cell surface glycoprotein involved in intercellular adhesion of rat hepatocytes. It has four extracellular immunoglobulin (Ig) domains, a transmembrane domain and a cytoplasmic domain and therefore is a member of the Ig supergene family. We have characterized multiple cDNAs of the C-CAM genes in rat intestine. Sequence analyses showed that rat intestine contained not only the previously reported L-form and S-form C-CAMs (renamed C-CAM1 and C-CAM2 respectively) but also a new isoform, C-CAM3. The C-CAM3 transcript codes for a polypeptide with a truncated C-terminus that lacks 65 amino acids from the previously reported C-CAM1 cytoplasmic domain. Unlike C-CAM1, C-CAM3 did not mediate cell adhesion when expressed in insect cells using the baculoviral expression system. Thus the extra 65 amino acids in the cytoplasmic domain of C-CAM1 are important for adhesion phenotype when expressed in insect cells. Although C-CAM1 and C-CAM2 are encoded by different genes, sequence analysis suggests that C-CAM3 is probably derived from alternative splicing of the C-CAM1 gene. To examine 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 unspliced intron which contained a stop codon immediately after the regular splice junction. As a result, translation of C-CAM3 terminates at the point where C-CAM1 and C-CAM3 sequences diverge. To investigate the expression of C-CAM1, C-CAM2 and C-CAM3 in different tissues, we used an RNAase-protection assay to simultaneously assess the levels of expression of these transcripts. Using total RNA prepared from various tissues, we showed that expression of C-CAM3 was tissue-specific, and the 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 indicate that C-CAM3 is probably an incomplete spliced product of C-CAM1 gene.
Cheung
P H
PH
Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Culic
O
O
Qiu
Y
Y
Earley
K
K
Thompson
N
N
Hixson
D C
DC
Lin
S H
SH
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D018076:DNA, Complementary; D007438:Introns; D008969:Molecular Sequence Data; D009036:Moths; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D015703:Antigens, CD; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D012333:RNA, Messenger; D000251:Adenosine Triphosphatases