Comprehensive Analysis of Expression and Function of 51 Sarco(endo)plasmic Reticulum Ca2+-ATPase Mutants Associated with Darier Disease

Abstract

We examined possible defects of sarco(endo)plasmic reticulum Ca catalyze Ca 2ϩ transport coupled with ATP hydrolysis (Fig. 1) and play an essential role in maintaining Ca 2ϩ homeostasis in the cytoplasm and endoplasmic reticulum lumen of cells (1-7). SERCAs have three cytoplasmic domains: phosphorylation (P), nucleotide binding (N), and actuator (A) and 10 transmembrane helices (M1-M10 or 11 in the SERCA2b isoform, M11). In the Ca 2ϩ transport cycle, the ATPase is activated by the binding of two Ca 2ϩ ions from the cytoplasm to the transport sites composed of M4, M5, M6, and M8 (E2 3 E1Ca 2 , step 1). Asp 351 in the P domain is then phosphorylated with MgATP to form the phosphorylated intermediate (EP) (step 2). During dephosphorylation of EP, the Ca 2ϩ ions are released into the lumen. In the detailed mechanism, the dephosphorylation process includes the conformational transition of EP associated with Ca 2ϩ release (step 3) and the subsequent hydrolysis of the acylphosphate bond (step 4).The three human SERCA genes encode SERCA isoforms (8 -10). Mutations in the SERCA2 gene (ATP2A2) and the resulting defects in the SERCA2b housekeeping isoform cause an autosomal dominant genetic skin disease, Darier disease (DD) (11,12). Over 100 mutations have been found with the DD pedigrees (11-24). They include many nonsense mutations, and also substitution and deletion mutations of amino acid residues. The mutations are located throughout the SERCA2b molecule and show no "hot spots" on the primary sequence. To understand how each of the substitution and deletion mutations affects SERCA2b protein, a limited number of mutations had been explored (9 by Ahn et al. (25), 10 by Dode et al. (23), 3 by Sato et al. (26) (a total of 20 because of overlap in Refs. 23 and 25)). To provide a comprehensive insight into the molecular basis of DD, as well as to understand the basis for each case of the DD pedigrees, it is necessary to analyze further the many unexplored substitution and deletion mutations. We therefore carried out in this study a comprehensive analysis of the expression and function of most of the DD causing substitution and deletion mutations reported, i.e. the 51 mutations shown in Fig. 2. Our results showed that most of the mutations (48 of the 51) cause severe defects in protein expression and/or Ca 2ϩ transport function. The loss of the transport function was ascribed to markedly reduced ATP hydrolysis or uncoupling from ATP hydrolysis. The remaining three mutations were exceptional in that they exhibited seemingly normal protein expression and

Darier’s disease: a calcium-signaling perspective

“…The potential role of Ca 2+ in the growth and differentiation of epithelial cells as well as keratinocytes is recognized [3,5,30]. Interestingly, the majority of SERCA2 mutations associated with classical DD reveal a reduction in the expression and activity of the pump [15][16][17][18][19][20]. Hence, an imbalance in cellular Ca 2+ signaling is an obvious readout of DD.…”

“…Since the identification of the gene responsible for DD, about 140 different mutations of SERCA2 have been evaluated with different degrees of phenotypic severity associated with the levels of expression and function of the pump. However, mutations rendering a state of SERCA2 haploinsufficiency are congruent with the classical DD phenotype [12,[15][16][17][18][19][20].…”

“…More recently in 2006, Hiroshi Suzuki and colleagues reported a comprehensive analysis of 51 SERCA mutants associated with DD. In their study, 48 of 51 mutants showed severe disruption of Ca 2+ homeostasis [20]. Although the skewed function of SERCA remains widely accepted disease etiology, the pathophysiological outcomes are quite pleiotropic.…”

Roles of Interaction between Actuator and Nucleotide Binding Domains of Sarco(endo)plasmic Reticulum Ca2+-ATPase as Revealed by Single and Swap Mutational Analyses of Serine 186 and Glutamate 439

“…2). The Darier disease-causing mutations of Ser 186 of SERCA2b, S186P and S186F also alter the kinetics of the EP processing and its impor-* This work was supported by a grant-in-aid for scientific research (B) ( tance as the residue in the immediate vicinity of TGES 184 has been pointed out (30,31). Notably also, Glu 439 is situated near the adenine binding pocket and its importance in the ATP binding and ATP-induced structural change have been shown (32,33 …”

Insights into Desmosome Biology from Inherited Human Skin Disease and Cardiocutaneous Syndromes

“…In vitro studies using Darier keratinocytes showed a decreased ER Ca 2 ϩ concentration (Dode et al, 2003;Foggia et al, 2006;Miyauchi et al, 2006;Pani et al, 2006). Upon Ca 2 ϩ depletion, misfolded proteins accumulated in the ER leading to induction of the unfolded protein response (UPR; Yoshida, 2007).…”