purification-endogenous-drosophila-transient-receptor-potential

Purification of Endogenous <em>Drosophila</em> Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...

Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center

The Eph receptor tyrosine kinase (RTK) family is the largest subfamily of RTKs playing critical roles in many developmental processes such as tissue patterning, neurogenesis and neuronal circuit formation, angiogenesis, etc. How the 14 Eph proteins, via their highly similar cytoplasmic domains, can transmit diverse and sometimes opposite cellular signals upon engaging ephrins is a major unresolved question. Here, we systematically investigated the bindings of each SAM domain of Eph receptors to the SAM domains from SHIP2 and Odin, and uncover a highly specific SAM-SAM interaction-mediated cytoplasmic Eph-effector binding pattern. Comparative X-ray crystallographic studies of several SAM-SAM heterodimer complexes, together with biochemical and cell biology experiments, not only revealed the exquisite specificity code governing Eph/effector interactions but also allowed us to identify SAMD5 as a new Eph binding partner. Finally, these Eph/effector SAM heterodimer structures can explain many Eph SAM mutations identified in patients suffering from cancers and other diseases.

Specific Eph receptor-cytoplasmic effector signaling mediated by SAM-SAM domain interactions.

Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca modulated CaM/TRP interaction is poorly understood. Here, we discover an unexpected, Ca-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an ∼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca-mediated feedback on the channel activity. Finally, we discover that TRPC4, the closest mammalian paralog of Drosophila TRP, adopts a similar CaM binding mode.

Weidi Chen

Shenzhen Key Laboratory for Neuronal Structural Biology,
Biomedical Research Institute,
Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute

Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Weidi Chen

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Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Shenzhen Key Laboratory for Neuronal Structural Biology,
Biomedical Research Institute,
Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute