Activation of certain phosphoinositidase-C-linked cell-surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and, thus, lead to Ins(1,4,5)P3-receptor down-regulation. In the current study we have sought to determine whether the ubiquitin/proteasome pathway is involved in this adaptive response. The data presented show (i) that activation of phosphoinositidase-C-linked receptors causes Ins(1,4,5)P3-receptor ubiquitination in a range of cell types (AR4-2J cells, INS-1 cells and rat cerebellar granule cells), (ii) that the Ins(1,4,5)P3-receptor down-regulation induced by activation of these receptors is blocked by proteasome inhibitors, (iii) that all known Ins(1,4,5)P3 receptors (types I, II and III) are substrates for ubiquitination, (iv) that ubiquitination occurs while Ins(1,4,5)P3 receptors are membrane-bound, (v) that Ins(1,4, 5)P3-receptor ubiquitination and down-regulation are stimulated only by those agonists that elevate Ins(1,4,5)P3 concentration persistently, and (vi) that a portion of cellular Ins(1,4,5)P3 receptors (those that are not type-I-receptor-associated) can be resistant to ubiquitination and degradation. In total these data indicate that the ubiquitin/proteasome pathway mediates Ins(1,4, 5)P3-receptor down-regulation and suggest that ubiquitination is stimulated by the binding of Ins(1,4,5)P3 to its receptor.

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...

Dimerization and phosphorylation of thyrotropin-releasing hormone (TRH) receptors was characterized using HEK293 and pituitary GHFT cells expressing epitope-tagged receptors. TRH receptors tagged with FLAG and hemagglutinin epitopes were co-precipitated only if they were co-expressed, and 10 -30% of receptors were isolated as hemagglutinin/FLAG-receptor dimers under basal conditions. The abundance of receptor dimers was increased when cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a dose-dependent manner. TRH increased phosphorylation of both receptor monomers and dimers, documented by incorporation of 32 P and an upshift in receptor mobility reversed by phosphatase treatment. The ability of TRH to increase receptor phosphorylation and dimerization did not depend on signal transduction, because it was not inhibited by the phospholipase C inhibitor U73122. Receptor phosphorylation required an agonist but was not blocked by the casein kinase II inhibitor apigenin, the protein kinase C inhibitor GF109203X, or expression of a dominant negative form of G protein-coupled receptor kinase 2. TRH receptors lacking most of the cytoplasmic carboxyl terminus formed dimers constitutively but failed to undergo agonist-induced dimerization and phosphorylation. TRH also increased phosphorylation and dimerization of TRH receptors expressed in GHFT pre-lactotroph cells.The TRH 1 receptor belongs to the superfamily of seventransmembrane-helix G protein-coupled receptors (GPCRs) and plays a key role in maintaining proper function of the thyroid gland (1, 2). Two subtypes of TRH receptors, termed type 1 and 2, have been identified (3-5). Although both receptor types are detected in various tissues at different levels (6 -8), the type 1 TRH receptor is primarily expressed in thyrotrophs and lactotrophs in the anterior pituitary gland, and its activation stimulates the secretion of TSH and prolactin, at least in part, by raising the intracellular calcium concentration (9, 10). The TRH receptor, once occupied by agonist, activates phospholipase C through G q/11 , leading to the formation of inositol 1,4,5-trisphosphate (InsP 3 ), which causes an elevation of intracellular calcium by mobilizing an InsP 3 -sensitive Ca 2ϩ store in the endoplasmic reticulum (10, 11).In conventional models, GPCRs have been thought to function as monomers that bind one molecule of ligand and then activate one heterotrimeric G protein to turn on the cognate signaling pathway (12-16). However, evidence indicating that GPCRs can form homo-or heterodimers, pairing with the same receptor type, different subtypes within the same receptor family, or even distinct classes of receptors, has begun to emerge. Receptor dimerization has been documented for a wide variety of GPCRs including the ␤ 2 -adrenergic receptor (17, 18), Ca 2ϩ -sensing receptor (19,20), muscarinic m3 receptor (21), ␥-aminobutyric acid GABA B receptor (...

These studies were designed to characterize ubiquitination of the G protein-coupled TRH receptor (TRHR). TRHRs and ubiquitin coprecipitated with antibodies to either receptor or ubiquitin in Chinese hamster ovary or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRHRs and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRHR coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRHRs were particularly heavily ubiquitinated. These results show that the proteasome participates in TRHR quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRHRs were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and nonpermissive temperatures, indicating that ligand-dependent ubiquitination of the receptor, or any other protein, is not necessary for TRHR signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.

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