Proteolytic processing of the C terminus of the alpha(1C) subunit of L-type calcium channels and the role of a proline-rich domain in membrane tethering of proteolytic fragments.
Although most L-type calcium channel alpha(1C) subunits isolated from heart or brain are approximately 190-kDa proteins that lack approximately 50 kDa of the C terminus, the C-terminal domain is present in intact cells. To test the hypothesis that the C terminus is processed but remains functionally associated with the channels, expressed, full-length alpha(1C) subunits were cleaved in vitro by chymotrypsin to generate a 190-kDa C-terminal truncated protein and C-terminal fragments of 30-56 kDa. These hydrophilic C-terminal fragments remained membrane-associated. A C-terminal proline-rich domain (PRD) was identified as the mediator of membrane association. The alpha(1C) PRD bound to SH3 domains in Src, Lyn, Hck, and the channel beta(2) subunit. Mutant alpha(1C) subunits lacking either approximately 50 kDa of the C terminus or the PRD produced increased barium currents through the channels, demonstrating that these domains participate in the previously described (Wei, X., Neely, a., Lacerda, A. E. Olcese, r., Stefani, E., Perez-Reyes, E., and Birnbaumer, L. (1994) J. Biol. Chem. 269, 1635-1640) inhibition of channel function by the C terminus.
Gerhardstein
B L
BL
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Gao
T
T
Bünemann
M
M
Puri
T S
TS
Adair
A
A
Ma
H
H
Hosey
M M
MM
D000818:Animals; D001464:Barium / Q000378:metabolism; D001665:Binding Sites; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004553:Electric Conductivity; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006352:Heart Ventricles / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011392:Proline; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018909:src Homology Domains
D020746:Calcium Channels, L-Type; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D001464:Barium; D011392:Proline; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases