Enkurin is a novel calmodulin and TRPC channel binding protein in sperm

Abstract

The TRPC cation channel family has been implicated in receptor- or phospholipase C (PLC)-mediated Ca2+ entry into animal cells. These channels are present in mammalian sperm and are assigned a role in ZP3-evoked Ca2+ influx that drives acrosome reactions. However, the mechanisms controlling channel activity and coupling Ca2+ entry through these channels to cellular responses are not well understood. A yeast two-hybrid screen was carried out to identify TRPC-interacting proteins that would be candidate regulators or effectors. We identified a novel protein, enkurin, that is expressed at high levels in the testis and vomeronasal organ and at lower levels in selected other tissues. Enkurin interacts with several TRPC proteins (TRPC1, TRPC2, TRPC5, but not TRPC3) and colocalizes with these channels in sperm. Three protein-protein interaction domains were identified in enkurin: a C-terminal region is essential for channel interaction; an IQ motif binds the Ca2+ sensor, calmodulin, in a Ca2+-dependent manner; and a proline-rich N-terminal region contains predicted ligand sequences for SH3 domain proteins, including the SH3 domain of the p85 regulatory subunit of 1-phosphatidylinositol-3-kinase. We suggest that enkurin is an adaptor that functions to localize a Ca2+ sensitive signal transduction machinery in sperm to a Ca2+-permeable ion channel.

Membrane translocation of TRPC6 channels and endothelial migration are regulated by calmodulin and PI3 kinase activation

“…PIP3 binding to a PH domain can induce conformational changes that affect protein function, or PIP3 binding can serve to colocalize proteins and regulate interactions such as oligomerization (31). On the other hand, TRPC6 might bind to PIP3 through a protein partner as has been reported for other TRPC proteins (32). Further studies are needed to determine if an adapter protein is required for TRPC6 binding to PIP3.…”

Regulating the acrosome reaction

“…It also binds TRPC1 and C5 (but not TRCP3), although those interaction sites have not yet been mapped (Sutton et al, 2004). This binding specificity corresponds to the functional and se- Fig.…”

“…The seven members of the TRPC family form Ca 2+ -conducting channels that are widely expressed in animal cells and contribute to PLC-dependent Ca 2+ entry (Montell, 2005;Putney, 2007). Several TRPC channels are expressed in sperm, with TRPC1, C2 and C5 are located in the anterior head where sperm interact with the zona pellucida and where acrosome reaction signaling occurs (Trevino et al, 2001;Jungnickel et al, 2001;Castellano et al, 2003;Sutton et al, 2004;Stamboulian et al, 2005). A substantial fraction of the ZP3-evoked Ca 2+ influx into mouse sperm is conducted by TRPC2, as a function-blocking antibody against the second extracellular loop of that channel inhibits both 80-85% of the sustained Ca 2+ i response and also blocks the acrosome reaction (Jungnickel et al, 2001).…”

“…Enkurin also contains an IQ motif that binds Ca 2+ -calmodulin, but not apocalmodulin, and therefore associates a Ca 2+ sensor with the inner face of a Ca 2+ -conducting ion channel (Sutton et al, 2004). Enkurin has the anticipated sequence characteristics of an adapter protein that binds SH3 and WW domain cargo to ZP3-activated TRPC channels, thereby assembling a signaling module at the site of ZP3 action.…”

Sperm channel diversity and functional multiplicity

“…STIM (Stromal Interaction Molecule) is a TRP-interacting protein that translocates from the ER to the plasma membrane to function either as the communication factor between the Ca 2þ -store and the plasma membrane or forming part of the channel itself . TRP channels can also be regulated by phosphatidylinositol biphosphate (PIP 2 ; Suh & Hille 2005) and by newly discovered regulatory proteins such as junctate (Stamboulian et al 2005) and enkurin (Sutton et al 2004) that bind specifically to certain TRP isoforms modulating their activity.…”