Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Alzayady, Kamil J.; Wagner, Larry E.; Chandrasekhar, Rahul; Monteagudo, Alina; Godiska, Ronald; Tall, Gregory G.; Joseph, Suresh K.; Yule, David I.

doi:10.1074/jbc.m113.502203

Cited by 42 publications

(68 citation statements)

References 59 publications

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“…Generation of Concatenated IP 3 R Constructs-R1 and R2 dimeric constructs were generated by designating IP 3 R cDNA encoding the corresponding isoforms as "head" and "tail" and modifying them appropriately using a QuikChange mutagenesis strategy as described before (39). R3 head subunits were generated by silently mutating NcoI sites in the rat R3 coding sequence (primers 1-6).…”

Section: Methodsmentioning

confidence: 99%

“…To generate IP 3 R dimers encoding homo-and heterodimeric receptors, the appropriate fragments were directly ligated between the two arms of pJAZZ Mamm linear vector to generate a construct encoding two IP 3 R subunits connected with a 14-amino acid linker within a single open reading frame. The tetrameric R1R1R1R2 construct was engineered by ligating, C to N terminus, subunits 1-3 of the R1R1R1R1 homotetramer and the R2 tail subunit, generated as described previously (39).…”

Section: Methodsmentioning

confidence: 99%

“…Native Tissues-The ubiquitous expression of IP 3 Rs is well established, with numerous studies unequivocally demonstrating that individual cell types express at least two if not all three isoforms (38,39). Co-immunoprecipitation experiments performed on lysates isolated from individual cell types or tissues have shown that these isoforms do associate (39,42,43,51).…”

Section: Ip 3 R Isoforms Oligomerize Forming Heterotetramers Inmentioning

confidence: 99%

“…Notably, most cell types express at least two subtypes (35)(36)(37). For instance, although the majority of neuronal IP 3 Rs are R1, most other peripheral cell types express R2 and R3 (38,39). Furthermore, numerous cross-linking (40,41), co-immunoprecipitation (39,42,43), and co-localization and immunostaining assays (44 -47) have supported the existence of heterotetrameric IP 3 R. Additionally, we demonstrated that homotetramers account for only a small proportion of IP 3 Rs in mouse pancreas (39).…”

mentioning

confidence: 99%

“…Studies in cultured cells or isolated tissues fail to account for the exact proportions of each subtype within a tetramer. To address this, we recently described a strategy whereby concatenated IP 3 R cDNA constructs were engineered to result in expression of tetrameric channels with defined composition (39). As proof of principle, we showed that concatenated homo-or heterodimers encoding R1 or R2 were stably expressed in DT40 triple IP 3 R knock-out (3KO) cells, dimerized to form tetrameric channels localized to the ER membrane, were functionally responsive to G q -coupled, G-protein-coupled receptor stimulation, and exhibited identical single channel activity to those expressed from monomeric constructs (39).…”

mentioning

confidence: 99%

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Unique Regulatory Properties of Heterotetrameric Inositol 1,4,5-Trisphosphate Receptors Revealed by Studying Concatenated Receptor Constructs

Chandrasekhar

Alzayady

Wagner

et al. 2016

Journal of Biological Chemistry

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were varied, we found that one subtype dictated the ATP regulatory properties of heteromers. We show that R2 monomers within a heterotetramer were both necessary and sufficient to dictate the ATP regulatory properties. Finally, the ATP-binding site B in R2 critical for ATP regulation was mutated and rendered non-functional to address questions relating to the stoichiometry of IP 3 R regulation. Two intact R2 monomers were sufficient to maintain ATP regulation in R2 homotetramers. In summary, we demonstrate that heterotetrameric IP 3 R do not necessarily behave as the sum of the constituent subunits, and these properties likely extend the versatility of IP 3 -induced Ca 2؉ signaling in cells expressing multiple IP 3 R isoforms.The importance of intracellular Ca 2ϩ as a second messenger is underscored by its distinctive ability to regulate a multitude of diverse cellular processes, including transcription, translation, secretion of fluids, muscle contraction, motility, fertilization, memory, apoptosis, and autophagy (1-8). This remarkable capacity to precisely and often simultaneously regulate cellular events is thought to be due, at least in part, to the highly sophisticated spatial and temporal control of intracellular [Ca 2ϩ ] by a complement of specialized proteins, collectively termed the "Ca 2ϩ signaling toolkit" (7). Simplistically, this "toolkit" includes several Ca 2ϩ influx and release channels whose activation brings about a rise in basal intracellular Ca 2ϩ concentration. In addition, cytosolic Ca 2ϩ buffers, pumps, and transporters function to reduce the intracellular Ca 2ϩ concentration by extrusion into stores or the extracellular space (9). Essential components of this toolkit are the members of the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptor (IP 3 R) 2 family. Stimulation of cell surface receptors by growth factors, hormones, and neurotransmitters results in Ca 2ϩ mobilization as a result of the generation of IP 3 , which subsequently binds to and activates IP 3 Rs (10, 11). There are three major subtypes of IP 3 Rs (R1, R2, and R3), encoded by a distinct gene (Itpr1, Itpr2, and Itpr3) (12, 13). These ϳ300-kDa monomeric proteins co-translationally oligomerize into ϳ1200-kDa tetrameric Ca 2ϩ release channels (14). The three subtypes share ϳ60 -70% sequence homology and are conventionally divided into three functional domains. At the extreme N terminus lies the conserved ligandbinding domain, which is composed of a suppressor domain (SD) and a ligand-binding core (15). This is followed by a large, less conserved intermediary regulatory domain that contains several putative sites for regulation by different molecules, including Ca 2ϩ (serving as a co-agonist), adenosine triphosphate (ATP), protein binding partners, and post-translational modifications (16). Finally, at the C terminus lies a six-transmembrane domain that, in addition to being critical for receptor oligomerization (17) and ER localization (18,19), contains the ion-conducting pore between transmembrane helice...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%