Calnexin and Calreticulin, ER Associated Modulators of Calcium Transport in the ER

Abstract

Calreticulin (CRT) and calnexin (CNX) are members of a family of endoplasmic reticulum (ER) chaperones that fold newly synthesized polypeptides. Aside from their role as foldases in the ER, our laboratory has shown that all members of this family of proteins modulate Ca2+ oscillations. In Xenopus oocytes and other cells, stimulation by G-protein and tyrosine coupled receptors results in Ca2+ release from the Inositol 1,4,5 trisphosphate receptor (IP3R) located in the ER. Following release, Ca2+ is re-sequestered into the ER by Ca2+ ATPases of the SERCA family. CRT and CNX overexpression inhibit Ca2+ oscillations when co-expressed with SERCA2b or when oocytes are treated with pyruvate malate to induce oscillations. By domain deletion mutagenesis of CRT we have determined that the N and P domains are necessary for the inhibition of Ca2+ oscillations. The mechanism of inhibition may involve a lectin-like interaction since mutagenesis of a lumenal asparagine to alanine in SERCA2b destroys the inhibitory effect. Coexpression of SERCA2a (which lacks the luminal asparagine) with either CRT or CNX does not inhibit Ca2+ oscillations, consistent with the notion that a lectin interaction may be involved. Unlike CRT, which is entirely lumenal, CNX has a cytosolic domain that is phosphorylated by multiple kinases. Mutagenesis of two PKC/PDK residues in CNX indicated that S562 supports phosphorylation. Expression of SERCA2b with a mutated CNX in S562 prevents the inhibition of Ca2+ oscillations suggesting that this residue serves as a phosphorylatable regulatory switch controlling the interaction of CNX with SERCA2b. Indeed, immunoprecipitations with a CNX specific antibody of oocytes treated with or without IP3 and preloaded with [γ-32P]-ATP demonstrated that S562 is phosphorylated at rest and dephosphorylated in response to IP3. Phosphorylation-mediated control of the interaction of CNX with SERCA2b is of significance since it suggests a bi-directional mode of communication between the Ca2+ signaling system and the folding machinery in the ER to maintain Ca2+ homeostasis in the organelle. The maintenance of Ca2+ homeostasis in the ER is then essential for protein folding.