A second isoform of chicken brush border myosin I contains a 29-residue inserted sequence that binds calmodulin.
Chicken brush border myosin I (CBB-MI) is a single-headed, nonfilamentous, myosin-like mechanoenzyme which, as isolated, has 3 mol of calmodulin (CAM) 'light chains' bound per mole of 119 kDa heavy chain. We have isolated a partial cDNA clone for CBB-MI that encodes the C-terminal approximately 35 kDa of the heavy chain. The sequence of this clone is identical to that of an authentic, near-full-length CBB-MI cDNA clone reported recently, except for an 87-bp/29-residue insertion occurring approximately 32 kDa from the C-terminus. This insert, which is probably generated by an alternate splicing event, is expressed in brush border as part of a message of the size predicted for the CBB-MI heavy chain, although the steady state level of this transcript is approximately 8-fold lower than for transcripts lacking the insert. 125I-CAM overlays of this cDNA clone (expressed as a trpE fusion protein in E. coli) indicate that it binds one more calmodulin than does a second cDNA clone that lacks the 29-residue insert. A synthetic peptide corresponding to the insert sequence binds tightly to CAM-Sepharose, demonstrates a shift and enhancement of fluorescence in the presence of CAM, and binds CAM in solution with a KD of 190 nM (in 100 mM KCl). We conclude that a second, low-abundance isoform of CBB-MI contains an additional (and possibly fourth) CAM binding site as a result of a 29-residue peptide that is inserted into the tail domain by an apparent alternate splicing event.
Halsall
D J
DJ
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Hammer
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D004247:DNA / Q000032:analysis; D005453:Fluorescence; D007536:Isomerism; D008871:Microvilli / Q000378:metabolism*; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D015879:Myosin Subfragments; D004247:DNA