A Carboxyl-terminal Region Important for the Expression and Targeting of the Skeletal Muscle Dihydropyridine Receptor

Catherine Proenza,Christina Wilkens,Nancy M Lorenzon,Kurt G Beam

doi:10.1074/jbc.m003389200

Catherine Proenza, Christina Wilkens + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m003389200

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Abstract

We have used the yeast two-hybrid technique and expression of truncated/mutated dihydropyridine receptors (DHPRs) to investigate whether the carboxyl tail of the DHPR is involved in targeting to junctions between the sarcolemma and sarcoplasmic reticulum in skeletal muscle. The carboxyl tail was extremely reactive in yeast two-hybrid library screens, with the reactivity residing in amino acids 1621-1647 and abolished by a point mutation (V1642D). Dysgenic myotubes were injected with cDNA encoding green fluorescent protein fused to the amino terminus of DHPRs truncated after either residue 1620 (Delta1621-1873) or residue 1542 (Delta1543-1873) or of full-length DHPRs with the V1642D mutation (V1642D). For either Delta1621-1873 or V1642D, the restoration of excitation-contraction coupling was reduced approximately 40%, and the number of functional DHPRs in the sarcolemma was reduced approximately 30%, compared with the wild-type DHPR. The restoration of excitation-contraction coupling and surface expression was more drastically reduced (by approximately 90 and approximately 55%, respectively) for Delta1543-1873. Fluorescence microscopy revealed that Delta1621-1873 and V1642D were concentrated in a longitudinally restricted region near the injected nucleus, whereas wild-type DHPRs were present relatively uniformly along the length of a myotube. The intensity of fluorescence was greatly reduced for Delta1543-1873, indicating a low level of protein expression. Thus, residues 1543-1647 appear to play a role in the biosynthetic processing, transport, and/or anchoring of DHPRs, with residues 1543-1620 being particularly important for expression.

Highlights

In skeletal muscle cells, electrical excitation of the sarcolemma causes Ca2ϩ to be released from intracellular stores within the sarcoplasmic reticulum, which in turn causes contraction
We have shown that the carboxyl tail of ␣1S is highly reactive in yeast two-hybrid screens of a skeletal muscle cDNA library, that this reactivity resides in residues 1621– 1647, and that the reactivity is abolished by the V1642D point mutation
We have shown that introduction of the V1642D mutation into the full-length dihydropyridine receptors (DHPRs) or truncation after residue 1620 reduces the fraction of dysgenic myotubes with restored EC coupling and lowers Qmax, suggesting a decreased surface expression of the altered DHPRs

Summary

Targeting of Dihydropyridine Receptors

DHPRs in dysgenic myotubes showed that either truncation or mutation of the carboxyl tail resulted in a decreased efficiency for restoration of EC coupling, a reduction of functional DHPRs expressed in the sarcolemma, and a reduced expression of DHPRs at sites longitudinally distant from the nucleus injected with cDNA. Taken together, these results suggest that residues. PRs in the sarcolemma, perhaps as a consequence of interaction with other proteins

EXPERIMENTAL PROCEDURES

RESULTS

Clone Restoration of ECC

DISCUSSION