Inositol 1,4,5-Trisphosphate Receptor Down-regulation Is Activated Directly by Inositol 1,4,5-Trisphosphate Binding

Abstract

When certain types of G-protein-coupled cell surface receptors (for example, M3 muscarinic receptors) are occupied by their cognate agonists, phosphoinositidase C (PIC) 1 is activated, phosphatidylinositol 4,5-bisphosphate is hydrolyzed and inositol 1,4,5-trisphosphate (InsP 3 ) and diacylglycerol are formed (1). InsP 3 is a second messenger that elicits calcium signals within cells that mediate many physiological processes (1, 2). The primary effect of InsP 3 is to trigger calcium release from the endoplasmic reticulum, thus raising cytoplasmic free calcium concentration ([Ca 2ϩ ] i ) (1, 2). This is achieved by interaction of InsP 3 with InsP 3 receptors, proteins that form tetrameric complexes in the endoplasmic reticulum membrane and that act as calcium channels (3, 4).Three types of InsP 3 receptor, namely, types I, II, and III, have been defined; they have similar sizes (2670 -2749 amino acids) and the same basic structure (3-7). For the type I InsP 3 receptor, which is the predominant type in neuronal cells (3-5), three domains have been defined: an InsP 3 -binding domain within the N-terminal 650 amino acids, a transmembrane or channel-forming domain close to the C terminus, and an intervening coupling domain (3,4,8). Several lines of evidence indicate that a conformational change occurs upon InsP 3 binding and that this is responsible for channel opening (3,4,8).The waning of cellular responses during persistent activation of cell surface receptors is a well documented phenomenon (9) and is evident for PIC-coupled receptors (10, 11). Such "desensitization" is mediated by several mechanisms, some of which occur acutely (within minutes), and some of which require long-term exposure to agonists (9 -11). One of the mechanisms by which cells adapt during long-term agonist exposure is by down-regulation of cell surface receptors, which is characterized by a decline in the cellular content of these proteins (9 -11). Remarkably, it has recently been found that InsP 3 receptors are also subject to down-regulation upon stimulation of PIC-linked cell surface receptors (12-16), providing a novel locus of adaptation. It has also been shown that InsP 3 receptor down-regulation can be induced by receptor-independent activation of . This phenomenon is seen with types I, II, and III InsP 3 receptors in a range of cell types (12-16). For example, stimulation of M3 muscarinic receptors in SH-SY5Y human neuroblastoma cells with carbachol (CCh), a metabolically stable analogue of acetylcholine, reduces type I InsP 3 receptor immunoreactivity by ϳ90%, with half maximal effect at 0.5-1 h (13,20). This reduction in InsP 3 receptor content is a specific process, as the other proteins are not simultaneously down-regulated (14 -16, 20). Moreover, the down-regulation is not related to changes in mRNA levels, but rather, it results from a profound acceleration of InsP 3 receptor degradation (13). The responsible proteolytic mechanism has yet to be defined but has been proposed to involve either calpain (20) or the ubiquitin/pr...

Ligand binding directly stimulates ubiquitination of the inositol 1,4,5-trisphosphate receptor

“…SH-SY5Y cells were grown as monolayers as previously described [15,32]. The generation and maintenance of SIns(1,4,5)P $ RHA and S∆Ins(1,4,5)P $ RHA cells, which are transfected SH-SY5Y cells stably expressing haemagglutinin (HA) epitope-tagged mouse cerebellum wild-type type I receptor [Ins(1,4,5)P…”

“…$ RHA] and binding-defective mutant type I receptor [∆Ins(1,4,5)P $ RHA] respectively, have been described [32]. The HA epitope tag (YPYDVPDYA) was introduced at the receptor C-terminus [32] ; the binding-defective mutant receptor lacked 37 residues (316-352) in the ligand-binding domain [33].…”

“…The HA epitope tag (YPYDVPDYA) was introduced at the receptor C-terminus [32] ; the binding-defective mutant receptor lacked 37 residues (316-352) in the ligand-binding domain [33]. Culture medium was changed every 3 days and was replaced 1 day before experiments.…”

“…To replace Lys-350 (K350) with arginine (R) in Ins-(1,4,5)P $ RHA, pcWIHA, which carries the cDNA insert encoding Ins(1,4,5)P $ RHA [32], was mutated ; A"$(( was changed to G in the codon for K350 (A"$('AA"$()) by PCR-based sitedirected mutagenesis. In brief, primers P1 (5h-ATGTCGTAAC-AACTCCGCCCCATTG-3h) and P2 (5h-CCAGAGAGTATAC-CATTCTTTCTTGCGCGTTTC-3h) were used to amplify the region between nt 743 of the vector [32] and 1394 of the insert, and primers P3 (5h-GAAACGCGCAAGAAAGAATGGTAT-ACTCTCTGG-3h) and P4 (5h-CTGCTTCTGCATGAAGCCA-AACTGC-3h) were used to amplify the region between nt 1362 and 2080 of the insert.…”

“…In brief, primers P1 (5h-ATGTCGTAAC-AACTCCGCCCCATTG-3h) and P2 (5h-CCAGAGAGTATAC-CATTCTTTCTTGCGCGTTTC-3h) were used to amplify the region between nt 743 of the vector [32] and 1394 of the insert, and primers P3 (5h-GAAACGCGCAAGAAAGAATGGTAT-ACTCTCTGG-3h) and P4 (5h-CTGCTTCTGCATGAAGCCA-AACTGC-3h) were used to amplify the region between nt 1362 and 2080 of the insert. Thus mismatched nucleotides (underlined in P2 and P3) were introduced into the 1589 bp and 718 bp fragments produced.…”