Subunit Oligomerization, and Topology of the Inositol 1,4,5-Trisphosphate Receptor

Abstract

The inositol 1,4,5-trisphosphate receptor (InsP 3 R) is a tetrameric assembly of highly conserved subunits that contain multiple membrane-spanning sequences in the C-terminal region of the protein. In studies aimed at investigating the oligomerization and transmembrane topology of the type-1 InsP 3 R, a series of membranespanning region truncation and deletion plasmids were constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to assemble into tetrameric structures. The topology of the membranespanning region truncations and the full-length receptor was determined by immunocytochemical analysis of transfected COS-1 cells using complete or selective permeabilization strategies. Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consistent with the current model for the organization of the InsP 3 R in the endoplasmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenable reagents to study receptor subunit oligomerization. Fractionation of soluble and membrane protein components revealed that the first two membrane-spanning regions were necessary for membrane targeting of the receptor. Sedimentation and immunoprecipitation experiments show that assembly of the receptor subunits was an additive process as the number of membrane-spanning regions increased. Immunoprecipitations from cells co-expressing the full-length receptor and carboxyl-terminal truncations reveal that constructs expressing the first two or more membranespanning domains were capable of co-assembling with the full-length receptor. Inclusion of the fifth membrane-spanning segment significantly enhanced the degree of oligomerization. Furthermore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membrane-spanning region deletion constructions that terminate with the receptor's 145 carboxyl-terminal amino acids were found to have enhanced assembly characteristics and implicate the carboxyl terminus as a determinant in oligomerization. Our results reveal a process of receptor assembly involving several distinct yet additive components and define the fifth and sixth membrane spanning regions as the key determinants in receptor oligomerization.Inositol 1,4,5-trisphosphate (InsP 3 ) 1 is a well known second messenger that plays a pivotal role in the regulated release of intracellular calcium. Plasma membrane receptor-coupled activation of G-protein or tyrosine kinase-induced hydrolysis of phosphotidylinositol 4,5-bisphosphate results in the production of InsP 3 and subsequent efflux of Ca 2ϩ from endoplasmic reticulum stores. InsP 3 -mediated calcium release has been implicated in numerous, very diverse cellular processes including the initiation/propagation of Ca 2ϩ waves, cell growth, secretion, fertilization, and development (1...

Topology of the Ca 2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1)

“…Comparable observations to those that we have made concerning the insertion of the first hairpin were made in a topological study of the inositol 1,4,5-trisphosphate receptor (IP 3 R) (37). If the protein was truncated from the C terminus, it became soluble before deletion of a sequence later deduced to be the first transmembrane sequence.…”

Role of the Sequence Surrounding Predicted Transmembrane Helix M4 in Membrane Association and Function of the Ca2+ Release Channel of Skeletal Muscle Sarcoplasmic Reticulum (Ryanodine Receptor Isoform 1)

“…Immunocytochemical Analysis of EGFP Orientation-Transfected MEF cells seeded on coverslips were fixed with 4% paraformaldehyde in PBS and, subsequently, permeabilized selectively or nonselectively with streptolysin-O or Triton X-100, according to methods described previously (18,19). In brief, fixed cells were washed and treated for 15 min with either 1% Triton X-100 at room temperature or 200 units/ml streptolysin-O, previously activated by a 5-min incubation on ice with 10 mM dithiothreitol in PBS.…”

“…SLO permeabilizes plasma membranes, but not intracellular membranes, and has been used successfully in several studies to identify the orientation of membrane sequences in intracellular membrane proteins (18,19). Fixed cells were treated with 1% Triton-X-100 to permeabilize both cellular and intracellular membranes or with SLO to permeabilize the plasma membrane selectively and then labeled sequentially with JL-8 and Alexa-568.…”

“…This result must be viewed cautiously, however, since some membrane sequences with weak topogenic function require support from a partner stop transfer TM sequence to complete the signal anchor stop transfer hairpin loop and achieve both integration into the membrane and proper orientation (9,10). The first TM sequence of the inositol 1,4,5-trisphosphate receptor (IP 3 R) (19) is a case in point; IP 3 R, truncated after its first TM sequence, was soluble, but truncated IP 3 R containing both the first and second TM sequences was targeted to the ER (19). The first and second TM sequences in IP 3 R are likely to correspond to the M5/M6 TM sequences in RyR and not to the M4 region in RyR.…”

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

“…The functional channel is formed co-translationally by the tetrameric association of four individual receptor subunits (9,10). Each subunit has a binding site for InsP 3 toward the N terminus formed by a cluster of positively charged amino acids thought to coordinate the negatively charged phosphate groups of InsP 3 (11,12).…”