Targeting of Inositol 1,4,5-Trisphosphate Receptors to the Endoplasmic Reticulum by Multiple Signals within Their Transmembrane Domains

Abstract

Most inositol 1,4,5-trisphosphate receptors (IP 3 R) are expressed in the endoplasmic reticulum (ER), where their precise distribution underlies the spatially complex Ca 2؉ signals evoked by extracellular stimuli. The signals that target IP 3 R to the ER or, less commonly, to other membranes are unknown. We expressed yellow fluorescent protein-tagged fragments of type 1 IP 3 R alone or fused with a plasma membrane protein to establish the determinants of ER targeting in COS-7 cells. By using a combination of confocal imaging and glycoprotein analyses, we demonstrated that any pair of the six transmembrane domains (TMD) linked by a luminal loop retains the protein within the ER, and when attached to a plasma membrane protein (ICAM-1), prevents it from reaching the medial Golgi. TMD1 or TMD2 alone were accumulated in mitochondria, whereas TMD5 and TMD6 were retained in ER, but were unable to prevent ICAM from reaching the plasma membrane. We conclude that IP 3 R are targeted to the ER membrane only after synthesis of TMDs 1 and 2, and that after co-translational insertion of the remaining TMDs, redundant retention signals present in any pair of TMD retain IP 3 R in the ER.Inositol 1,4,5-trisphosphate receptors (IP 3 R) 1 are intracellular Ca 2ϩ channels that mediate the release of Ca 2ϩ from intracellular stores evoked by receptors that stimulate IP 3 formation (1). Their close relatives, ryanodine receptors (RyR), are another family of intracellular Ca 2ϩ channels with a more restricted distribution and different modes of regulation (2). There are three closely related subtypes (1-3) of mammalian IP 3 R, and although they and their splice variants differ subtly in some aspects of their regulation (3), their structures are likely to be similar (4). All IP 3 R are tetrameric, and each subunit, which may be identical or different within a receptor (3, 5, 6), has a cytosolic IP 3 -binding domain toward the Nterminal (7), separated from a channel toward the C-terminal by a large "modulatory domain" (8). Topology predictions and direct evidence (9, 10) suggest the existence of six transmembrane domains (TMD) toward the C-terminal of each IP 3 R subunit. The last two TMD, together with the intervening loop from each of the four subunits, form the pore (11). Although the transmembrane topology of RyR subunits is unresolved, they are known to have an even number of TMD (probably six or eight) (12) and, as with IP 3 R, the last pair of TMD form the pore (13); both the N and C termini are cytosolic. For both IP 3 R and RyR, the last pair of TMD, together with ϳ25 conserved residues lying ϳ35 residues downstream of them, are also major determinants of assembly of the tetrameric protein (10, 14, 15).Most IP 3 R are expressed in the ER membrane (16 -19), but IP 3 has also been reported to stimulate Ca 2ϩ release from the nuclear envelope (20 -22), nucleoplasmic reticulum (23), Golgi (24), and secretory vesicles (25), although the latter has been challenged (26). IP 3 R are also expressed in the plasma membrane of some cells (2...

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

“…Membrane Localization and Tetrameric Assembly-As transmembrane proteins, it is reported that IP 3 Rs are cotranslationally targeted first to membranes of the endoplasmic reticulum (ER) where they are either retained as permanent ER resident proteins or are trafficked to other organelles, such as the Golgi apparatus or plasma membrane (55). To determine whether concatenated dimers were properly targeted and retained in membranes, subcellular fractionation was performed using cells expressing IP 3 R1 and R1R1 as a representative of dimeric IP 3 R constructs.…”

IP3 Receptors: Toward Understanding Their Activation

“…If we are to understand how Ca 2þ functions as a ubiquitous intracellular messenger, we must explain how IP 3 -evoked Ca 2þ signals grow from the opening of a single IP 3 R to much larger events. That explanation depends, ultimately, on putting IP 3 R into appropriate places within the cell, and on the interactions between IP 3 and Ca 2þ in regulating the opening of IP 3 R. In recent reviews (Taylor et al 2009a;Taylor et al 2009b) and original reports, we have described how IP 3 R are co-translationally targeted to the ER and then retained there by sequences within their transmembrane domains (TMD) (Parker et al 2004;Pantazaka and Taylor 2010). We have also suggested that within the ER, IP 3 causes IP 3 R to assemble into small clusters within which their regulation by both IP 3 and Ca 2þ is retuned to facilitate the Ca 2þ -mediated recruitment of IP 3 R activity by an active neighbor ).…”

Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

“…The C terminus of each subunit is postulated to span intracellular membranes six times and forms a single cation-selective pore (13,14). In addition, this region signals retention of the protein to the endoplasmic reticulum (15,16). Although the InsP 3 -binding pocket and channel pore are highly conserved between InsP 3 R family members, the intervening sequence between the binding region and pore is more divergent and consists of the so-called "regulatory and coupling" or "modulatory" domain.…”