CLONING OF NOVEL KINECTIN SPLICE VARIANTS WITH ALTERNATIVE C-TERMINI - STRUCTURE, DISTRIBUTION AND EVOLUTION OF MOUSE KINECTIN

The analysis of cDNA clones encoding novel variant forms of mouse kine ctin, an endoplasmic reticulum (ER)-bound receptor for the motor prote in kinesin, is reported. Kinesin and cytoplasmic dynein are involved i n mediating the anterograde and retrograde movements of intracellular vesicles along the microtubule network. The amino acid sequence deduce d from kinectin cDNA isolated from mouse spleen cell and testis librar ies revealed a long signal peptide or transmembrane sequence, and a 32 8 amino acid residue globular N-terminal domain adjacent to a much lar ger 858-999-residue C-terminal coiled-coil rod domain. The C-terminal domain was composed of 18 coiled-coil regions formed from multiple con tiguous heptad repeats which undergo alternative splicing as evidenced by the presence of at least five small (23-33 amino acid residue) ins ertion sequences scattered throughout. The inserts are present in any one of a number of combinations, generating an array of novel kinectin variants. insert 5 contains a termination codon, producing a C-termin us that is highly homologous to that of human kinectin. Three out of f ive mouse kinectin clones lack insert 5, generating a novel eleven ami no acid C-terminus encoded by sequence that extends past the insertion site. The existence of alternative C-termini may have functional rele vance given that the C-termini are exposed for interaction with kinesi n, whereas the globular N-terminus is embedded in the ER membrane. Alt ernative C-termini represent candidate modifications that could determ ine specificity of binding to kinesin or cytoplasmic dynein, and the s witching of directionality of movement. The cDNA hybridized to 4.5 kb transcripts expressed in all mouse cell lines and tissues examined, wh ich provides the first indication that the kinectins are very widely d istributed. Mouse kinectin is 42% similar over a 203 amino acid region to the chicken extracellular cardiac morphogen ES/130, whose canine h omologue containing an inserted sequence of 10 amino acids repeated 54 times in tandem, is a ribosome receptor expressed on the ER. Mouse ki nectin shares 64 and 83% identity, respectively, with its M(r) 160000 chicken and human kinectin homologues, There is a two-fold molar exces s of kinectin over kinesin in unextracted vesicles, suggesting that ki nectin might be a dimer. The electrostatic properties of the coiled-co il region of mouse kinectin, together with the relative frequencies of residues in particular positions within the heptad repeats support th is notion.