Regulation of store-operated calcium entry by alternative splicing of STIM2 [electronic resource]

Store-operated calcium entry (SOCE) is a key calcium signaling pathway present in most cell types. The pathway is activated in response to signaling events, such as the activation of phospholipase C coupled receptors on the plasma membrane, that deplete the endoplasmic reticulum (ER) Ca2+-stores. Depletion of these stores is sensed by the STIM proteins, STIM1 and STIM2, residing on the ER membrane. Upon store-depletion, the dissociation of Ca2+ from their luminal domains triggers conformational changes in STIM proteins and leads to their accumulation at regions of close apposition of the ER and plasma membrane (PM). At these ER-PM junctions, STIMs directly bind and activate Orai calcium channels in the plasma membrane, leading to an influx of Ca2+. While STIM1 is thought to respond to higher levels of store-depletion and mediate responses to receptor signaling, STIM2 can be activated by even mild store-depletion and is thought to play a role in maintaining Ca2+ homeostasis. Ca2+ signaling through SOCE regulates several downstream responses such as transcriptional regulation, secretion, and cellular motility. Perturbations in SOCE are linked with defects in a number of tissue types, including defects in immune function, muscle development and function, platelet activation, skin homeostasis among others. The amplitude and dynamics of SOCE are important determinants of Ca2+ dependent downstream responses, and thus critical to maintaining these physiological functions. However, while the basic mechanism of SOCE activation by STIM-Orai coupling is well understood, mechanisms that modulate the amplitude and dynamics of SOCE are less clear. In recent years, a number of such modulatory mechanisms have been proposed, including transcriptional regulation of STIM proteins, post-translational modifications, and the binding of accessory proteins. These mechanisms produce quantitative changes in the activity of STIM proteins, but without altering their fundamental role as activators of Orai channels. A largely unexplored mechanism with the potential to qualitatively alter STIM function is alternative splicing. Both STIM1 and STIM2 are multi-exonal proteins, and hence are expected to form multiple splice isoforms. However, the alternative splicing of STIM proteins and its role in the regulation of SOCE has not been fully explored so far. In this thesis, I explore the role of a novel STIM2 splice isoform, named STIM2 [beta] (the conventional isoform being referred to as STIM2 [alpha]), in regulating SOCE. STIM2 [beta] is formed by the in-frame splicing of exon 9 of the STIM2 genomic locus. This splicing event leads to the insertion of 8-amino acids, referred to as the 2 [beta] insert, in the middle of STIM2's key Orai binding domain. In contrast to all known STIM isoforms, STIM2 [beta] was found to inhibit SOCE mediated by Orai1 channels. In addition, STIM2 [beta] acts as a negative regulator of cytosolic and ER Ca2+ levels. These functional changes suggest that the alternative splicing qualitatively changes STIM2 function and converts it from an activator to an inhibitor of SOCE. Analysis of STIM2 [beta] function, using FRET and puncta formation assays, revealed that the 2 [beta] insert disrupts STIM2 [beta] binding to Orai1. However, STIM2 [beta] maintains the ability to heterodimerize with STIM2 [alpha] and STIM1, and is tethered to Orai channels as part of these heterodimers. Mutations in the 2 [beta] insert were found to significantly reduce the ability of STIM2 [beta] to inhibit SOCE, indicating that the 2[beta] insert sequence plays a crucial role in the inhibitory mechanism. Analysis of STIM2 [beta] mutants and Orai1-STIM2 [beta] chimeras suggested that STIM2 [beta] actively inhibits Orai1 channels through a sequence-specific interaction. STIM2 [beta] thus functions as a store-operated inhibitor of Orai1 channels. These findings reveal a novel modulatory mechanism for modulating the amplitudes and dynamics of SOCE by regulating STIM2 splicing. The thesis begins with an introductory chapter discussing the function and key mechanistic aspects of Ca2+ signaling and SOCE. Chapter 2 describes the discovery and mechanistic studies of STIM2 [beta]. Chapter 3 provides a discussion of some of the key remaining questions regarding the regulation of SOCE by STIM2 [beta] and also describes some experimental approaches to answer them.

Keywords: Biochemistry.
Advisor: Lewis Richard (Richard Sheridan)
Ferrell James Ellsworth advisor. Thesis advisor
Krasnow Mark