A second isoform of chicken brush border myosin I contains a 29-residue inserted sequence that binds calmodulin

FEBS Lett. 1990 Jul 2;267(1):126-30. doi: 10.1016/0014-5793(90)80305-3.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Calmodulin / genetics
  • Calmodulin / metabolism*
  • Cattle
  • Chickens
  • DNA / analysis
  • Fluorescence
  • Isomerism
  • Microvilli / metabolism*
  • Molecular Sequence Data
  • Myosin Subfragments / genetics
  • Myosin Subfragments / metabolism*

Substances

  • Calmodulin
  • Myosin Subfragments
  • DNA