Regulation of NMDA-type glutamate receptors and MDR1 by two members of the EF-hand protein family Public Deposited


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  • EF-hand proteins are a conserved family of proteins that are modulated by divalent cations and regulate diverse cellular activities. In the present study we characterized the molecular determinants of myosin regulatory light chain (RLC) interaction with several subunits of the N-methyl-D-aspartate (NMDA)-type glutamate receptor. Myosin RLC is an EF-hand protein that is traditionally considered an integral component of the myosin II complex. We show that this light chain is important for trafficking of NMDA receptors and that this function is likely independent of the myosin II complex. In addition, we studied the interaction of myosin RLC with a second non-myosin target, multi-drug resistance protein 1 (MDR1) also known as P-glycoprotein. Finally, we characterized a previously undescribed calcium-dependent calmodulin binding site on the NMDAR 2A (NR2A) subunit of the NMDA receptor. Calmodulin is structurally-related to myosin RLC and also considered an EF-hand protein. Myosin II motors are hexameric complexes containing two heavy chains that each bind a pair of light chains: one essential light chain and one RLC. Alternative binding partners have been described for both light chains of myosin II raising the possibility that myosin RLC, like other EF-hand proteins, may adopt conformations that can be distinguished from conventional myosin RLC-heavy chain interactions. In this study, we mapped the myosin RLC binding site to a 30-37 amino acid region of the C-termini of NR1 and NR2 subunits. Myosin RLC-NMDA receptor subunit interactions could be distinguished from the prototypical interaction of myosin RLC with the neck region of non-muscle myosin II-B heavy chain. NMDA-myosin RLC interactions were maintained in the absence of the fourth EF-hand domain and did not require the addition of magnesium. We report that sequence similarity in the "GxxxR" portion of the incomplete IQ2 motif found in nonmuscle myosin II heavy chain isoforms likely contributes to the recognition of NR2A as a non-myosin target of the myosin RLC. We report that myosin RLC-NR2A interactions likely occur in the Golgi complex and this interaction is important in forward trafficking of NR1/NR2A receptors. We suggest that a role for myosin RLC in protein trafficking in polarized cells is distinct from the typical interaction of myosin RLC as a component of the myosin II complex. Like glutamate receptor subunits, MDR1 is also a membrane-bound protein expressed in polarized cells. MDR1 is a drug efflux transporter that consists of two homologous halves, each consisting of six membrane-spanning domains plus a nucleotide binding domain linked by an intracellular linker region. The linker region of MDR1 is a determinant of cell surface expression and directly interacts with intracellular cytoskeletal, regulatory, and motor proteins. A previous study documented a direct interaction between myosin RLC and the linker region of a related ABC transporter, bile salt export protein (BSEP), and established a role for myosin in BSEP trafficking. We found that myosin RLC interacts with MDR1 via the amino terminal of the light chain as observed with NMDA receptor subunits. This interaction of myosin RLC with both of its binding partners (MDR1 and NMDA receptor subunits) is decreased upon phosphorylation of specific residues in the amino terminal of the light chain. We used Madin-Darby canine kidney (MDCK) cells stably expressing MDR1 (MDCKII-MDR1) as a model system to study the functional consequences of perturbing the phosphorylation state of myosin RLC in intact cells. Treatment of polarized MDCKII-MDR1 monolayers with ML-7, a pharmacologic inhibitor of myosin light chain kinase, increased the permeability of [³H]-digoxin (a well-known substrate of MDR1) and decreased apical expression of MDR1 in MDCKII-MDR1 cells. The combination of NR1 splice variants and NR2 subunits imparts differing physiological and pharmacological properties on NMDA receptor assemblies. The NR2 C-termini of NMDA receptors are approximately 600 amino acids long and the middle region of NR2A C-terminus (NR2A (875-1029)) bears only 29% sequence similarity with the corresponding region in the NR2B subunit. We used a proteomics approach to uncover proteins that may interact with this region from mouse brain homogenates both in the presence and absence of calcium. Calmodulin was found to interact with NR2A (875-1029) in a calcium-dependent manner. The binding affinity of calmodulin for the NR2A subunit was found to be 5.2 ± 2.4 nM, which is comparable to the affinity of a previously described binding site of calmodulin on the NR1 subunit. We found that tryptophan at position W1014 in NR2A C-terminal is critical for interaction with calmodulin. We also confirm in our studies that calmodulin is not a binding partner of the NR2B subunit of the NMDA receptor. Together our studies provide insight to the interaction of two EF-hand proteins with target proteins. For the first time we provide a functional role of myosin RLC in trafficking of NMDA receptors to the plasma membrane. We also show that myosin RLC can influence cell surface expression of MDR1 and as a consequence alter the transport properties of MDR1 as a drug efflux transporter. This study will contribute to our understanding of the mechanisms that underlie increased expression of MDR proteins associated with drug refractory conditions. In addition, we characterized a novel calmodulin binding site on the NR2A subunit of the NMDA receptor. This previously undescribed calmodulin binding site within the NR2A C-terminus potentially highlights an important distinction between Ca²⁺/calmodulin regulation of NR2A versus NR2B containing complexes.
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