Down-regulation of types I, II and III inositol 1,4,5-trisphosphate receptors is mediated by the ubiquitin/proteasome pathway

Abstract

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.

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

“…This down-regulation decreases the sensitivity of Ca# + stores to Ins(1,4,5)P $ [12,18], is due to accelerated receptor proteolysis [14,15], has a half-time of 0.5-2 h [14][15][16][17][18], can decrease Ins(1,4,5)P $ R levels by as much as 90 % [14,15], is evident with all three receptor types [15,17,18], and occurs in a variety of cells in itro [14][15][16][17][18] and in i o [19]. Although the mechanism of Ins(1,4,5)P $ R proteolysis has yet to be defined, it might be executed through the ubiquitin\ proteasome pathway [17][18][19][20], a pathway crucial to basal and regulated degradation of many cytosolic, nuclear, and membrane proteins [21][22][23][24][25].…”

“…Thus ubiquitin conjugation has a central role, and seems to be the rate-limiting step, in directing proteins towards proteasomal degradation [21,26]. Several mechanisms [28][29][30][31] have been proposed as the stimulus or ' degradation signal ' for ubiquitination but no information is yet available on what activates Ins(1,4,5)P $ R ubiquitination, other than indirect evidence that Ins(1,4,5)P $ is the trigger ; this comes from studies showing that Ins(1,4,5)P $ R ubiquitination is seen only with those agonists that persistently elevate Ins(1,4,5)P $ concentration [20].…”

“…Cells were grown in dishes 10 cm in diameter to confluence and, before exposure to agonist, were preincubated for 2 h with Nacetyl-Leu-Leu-norleucinal (ALLN), a potent proteasome inhibitor [34] that leads to the accumulation of ubiquitinated proteins within cells [17,20]. Agonist-stimulated or control cells were then disrupted for 20 min in ice-cold lysis buffer [150 mM NaCl\50 mM Tris base\1 mM EDTA\1 % (v\v) Triton X-100\0.2 mM PMSF\1 mM dithiothreitol\10 µM leupeptin\ 10 µM pepstatin\0.2 µM soybean trypsin inhibitor (pH 8.0) ; 1 ml per dish of cells] and were centrifuged (14 000 g for 10 min at 4 mC).…”

“…The immunoprecipitates were then washed four times with 1 ml of lysis buffer and were finally resuspended in 80 µl of gel-loading buffer [100 mM Tris\HCl (pH 6.8)\200 mM dithiothreitol\4 % (w\v) SDS\0.2 % Bromophenol Blue\20 % (v\v) glycerol]. Samples and prestained molecular mass markers were then separated by SDS\PAGE [5 % (w\v) gel], transferred to nitrocellulose membranes and probed with CT1, HA11 or polyclonal anti-ubiquitin as described [20]. Immunoreactivity was detected by enhanced chemiluminescence (Pierce) and X-ray film [15,20], and was quantified with Quantity One software (Bio-Rad), using the range in which band intensity was linearly related to the quantity of ubiquitinated receptors loaded.…”

“…Samples and prestained molecular mass markers were then separated by SDS\PAGE [5 % (w\v) gel], transferred to nitrocellulose membranes and probed with CT1, HA11 or polyclonal anti-ubiquitin as described [20]. Immunoreactivity was detected by enhanced chemiluminescence (Pierce) and X-ray film [15,20], and was quantified with Quantity One software (Bio-Rad), using the range in which band intensity was linearly related to the quantity of ubiquitinated receptors loaded. Molecular mass values and quantified chemiluminescence are expressed as means (n l 3) or meanspS.E.M.…”