Multiple Effects of SERCA2b Mutations Associated with Darier's Disease

Abstract

Darier's disease (DD) is an autosomal dominant disorder caused by mutations in the ATP2A2 gene, encoding sarco/endoplasmic reticulum Ca 2؉ -ATPase pump type 2b isoform (SERCA2b). Although >100 mutations in the ATP2A2 gene were identified, no apparent relation between genotype/phenotype emerged. In this work, we analyzed 12 DD-associated mutations from all of the regions of SERCA2b to study the underlying pathologic mechanism of DD and to elucidate the role of dimerization in SERCA2b activity. Most mutations markedly affected protein expression, partially because of enhanced proteasome-mediated degradation. All of the mutants showed lower activity than the wild type pump. Notably, several mutants that cause relatively severe phenotype of DD inhibited the activity of the endogenous and the co-expressed wild type SERCA2b. Importantly, these effects were not attributed to changes in passive Ca 2؉ leak, inositol 1,4,5-trisphosphate receptor activity, or sensitivity to inositol 1,4,5-trisphosphate. Rather, co-immunoprecipitation experiments showed that SERCA2b monomers interact to influence the activity of each other. These findings reveal multiple molecular mechanisms to account for the plethora of pathologic states observed in DD and provide the first evidence for the importance of SERCA2b dimerization in pump function in vivo.Darier's disease (DD, 1 OMIM 124200), also known as keratosis follicularis, is an autosomal dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization (1). DD is also associated with an increased prevalence of neuropsychiatric disorders including bipolar disorders, schizophrenia, epilepsy, and mental retardation (1). Recently, mutations in the ATP2A2 gene encoding the sarco/endoplasmic reticulum Ca 2ϩ -ATPase pump type 2b isoform (SERCA2b) have been identified as a cause of DD (2). SERCA2b is a housekeeping protein, which is expressed ubiquitously and is responsible for Ca 2ϩ uptake into the ER, including the agonist-mobilizable Ca 2ϩ pool (3, 4). By determining ER Ca 2ϩ load, the SERCA2 pumps modulate several parameters of the Ca 2ϩ signal. In resting cells, the SERCA2 pump provides the bulk of the dynamic Ca 2ϩ -buffering capacity to prevent large fluctuation in [Ca 2ϩ ] i (5). In stimulated cells, SERCA2 controls the activity of the store-operated Ca 2ϩ influx channel by preventing accumulation of Ca 2ϩ at the mouth of the channels and their inhibition by [Ca 2ϩ ] i (6). SERCA2b reloads the ER with Ca 2ϩ at the end of cell stimulation (5) and between Ca 2ϩ spikes to determine the frequency of Ca 2ϩ oscillations (7), and to control the diverse cellular functions regulated by [Ca 2ϩ ] i and Ca 2ϩ oscillations (8, 9). Alteration in SERCA2 pump function by overexpression in Chinese hamster ovary cells (10) or partial deletion in mice (7) lead to adaptation of the Ca 2ϩ signaling machinery and Ca 2ϩ -regulated cell functions that translates to nearly normal physiological response in the majority of organs and tissues. This co...

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

“…Thus, cellular Ca 2ϩ transport function must be considerably disrupted by these mutations (in total 48) to result in an increase in cytoplasmic Ca 2ϩ and a decrease in endoplasmic reticulum lumenal Ca 2ϩ , hence DD. This is consistent with notions (23,25) that DD is caused by haploinsufficiency in Ca 2ϩ homeostasis or by a possible intermolecular interaction of the defective mutant with the wild type, which may possibly inhibit the function of the wild type or cause its protein degradation. On the other hand, exceptions among the 51 DD mutants were found in the three mutants I274V, L321F, and M719I as they exhibited seemingly normal protein expression and Ca 2ϩ transport function coupled with ATP hydrolysis (Fig.…”

“…Mutations N767S, A803T, G807R, and V843F, Causing Uncoupling-The DD mutants N767S on M5 and V843F on M7 at or near the high-affinity Ca 2ϩ -binding sites, and A803T and G807R on the cytoplasmic side of M6 have never been explored except V843F, for which Ca 2ϩ transport was previously reported to be significantly slowed (25). We found here that these mutations cause complete loss (A803T and V843F) or significant reduction (N767S and G807R) of Ca 2ϩ -transport activity due to strong or partial uncoupling from ATP hydrolysis (Fig.…”

“…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)).…”

Up-Regulation of Transient Receptor Potential Canonical 1 (TRPC1) following Sarco(endo)plasmic Reticulum Ca2+ATPase 2 Gene Silencing Promotes Cell Survival: A Potential Role for TRPC1 in Darier's Disease

“…It has been hypothesized that a single copy of the gene is insufficient to encode adequate amount of SERCA protein, and significant changes in cellular Ca 2ϩ regulation and cell growth are seen in these cells (Sakuntabhai et al, 1999;Ahn et al, 2003;Foggia et al, 2006). To examine the possible consequences of SERCA2 hypofunction, we knocked down SERCA2 in HaCaT cells using adenovirus encoding either SERCA2-siRNA or control-siRNA.…”

“…However, of the multiple isoforms of SERCA1, -2, or -3, only SERCA2b is expressed in keratinocytes (Lytton and MacLennan, 1988;Ruiz-Perez et al, 1999). The mutated SERCA2 fails to sequester cytosolic Ca 2ϩ into the endoplasmic reticulum (ER) lumen, thereby disturbing the otherwise normal Ca 2ϩ homeostasis circuitry within the cells (Zhao et al, 2001;Ahn et al, 2003). Although oral retinoids, such as isotretinoin, have been shown to reduce hyperkeratosis (Burge and Wilkinson, 1992), the mechanism behind dysfunction of SERCA2 resulting in keratosis remains elusive.…”