Inositol 1,4,5-trisphosphate receptors in endocrine cells: localization and association in hetero- and homotetramers.

Abstract

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular calcium channel involved in coupling cell membrane receptors to calcium signal transduction pathways within cells including endocrine cells. Several isoforms (I, II, and III) of IP3Rs have been identified, which are encoded by separate genes, and are expressed in many tissues with differing patterns of cellular expression. We have generated specific affinity-purified polyclonal anti-peptide antibodies to each of the three isoforms. Western blot analysis of RINm5F and ATt2O cells shows high levels of endogenously expressed type I and type III IP3R, but undetectable levels of type II. Immunofluorescence studies revealed an endoplasmic reticulum-like pattern similar to BiP, an ER marker. In contrast with previous claims, both type I and type III IP3Rs were absent from the secretory granules of ATt2O cells. Western blots of sucrose gradients and gel filtration probed with antibodies to either type I or type III showed a molecular weight of greater than 1,000 kDa consistent with a tetrameric structure. Co-immunoprecipitation experiments indicated that most of the receptors were present as heterotetramers. Homotetramers were identified for the type III IP3R; however, type I homotetramers were undetectable. These data suggest that molecular association of IP3Rs into heterotetrameric forms can contribute to the complexity of the regulation of Ca2' release from ER by IP3Rs within cells.

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

“…The functional impact of the co-expression of multiple IP 3 R sub-types in an individual cell may simply reflect redundancy or, alternatively, a means of fine-tuning the Ca 2ϩ signal to accomplish a particular physiological specialization (41,42,49). Added complexity has been suggested by several studies, performed mainly using cultured cell lines, demonstrating that IP 3 R subtypes form both homo-and heterotetrameric channels, although the relative levels of the assembled channel have not generally been established (14,16,18). To verify that heterotetrameric channels are assembled in native tissues, co-immunoprecipitation experiments were performed with lysates from the pancreas, parotid, and spleen.…”

“…Briefly, to make the head subunit, IP 3 R2 cDNA in pcDNA3.1(ϩ) vector was modified so that all NcoI sites in the coding region were silently mutated (primers [15][16][17][18]. A glycine codon (GGC) was inserted after the initiation methionine to create a new NcoI site and a Kozak sequence in the context of the start codon (primer 19).…”

“…The activity of individual homotetramers has been interrogated using single channel electrophysiology or fluorescence-based experiments providing fundamental information regarding the binding of IP 3 , together with the gating and regulation of the activity of individual IP 3 R subtypes (19,25,35,45). However, because virtually all cells express a complement of at least two IP 3 R subtypes, it is predicted that most cells express a combination of homo-and heterotetrameric channels (13,14,18). Assuming IP 3 R subunit oligomerization to form tetramers is a random process and follows a binomial distribution, co-expression of multiple IP 3 R subtypes would be predicted to lead to the formation of homomeric as well as heteromeric channels with many possible subunit (homo and hetero) combinations.…”

Protein kinase C phosphorylates the inositol 1,4,5-trisphosphate receptor type 2 and decreases the mobilization of Ca2+in pancreatoma AR4-2J cells

“…IP 3 Rs associate in tetrameric structures that form functional Ca 2C channels. Individual cell types can express more than one IP 3 R subtype, which may form different populations of homo-and hetero-tetrameric channels (Joseph et al 1995, Monkawa et al 1995, Wojcikiewicz & He 1995, Nucifora et al 1996. The relatively poor homology between the regulatory domains of the three IP 3 Rs subtypes is likely to confer specific pharmacological and functional properties to each subtype.…”

Individual Subtypes of Enteroendocrine Cells in the Mouse Small Intestine Exhibit Unique Patterns of Inositol 1,4,5-trisphosphate Receptor Expression

“…Type 1, 2, and 3 receptors (IP3R-1, IP3R-2, and IP3R-3, respectively) are encoded by different genes and exhibit a high degree of overall sequence homology. IP3Rs assemble into homo-or heterotetrameric complexes (Nucifora et al 1996). Because each isoform exhibits unique regulatory properties, complexes of different IP3Rs display different calcium-gating properties.…”