The T-cell costimulatory receptors, CD28 and the inducible costimulator (ICOS), are required for the generation of follicular B helper T cells (T FH) and germinal center (GC) reaction. A common signal transducer used by CD28 and ICOS is the phosphoinositide 3-kinase (PI3K). Although it is known that CD28-mediated PI3K activation is dispensable for GC reaction, the role of ICOS-driven PI3K signaling has not been defined. We show here that knock-in mice that selectively lost the ability to activate PI3K through ICOS had severe defects in T FH generation, GC reaction, antibody class switch, and antibody affinity maturation. In preactivated CD4 ؉ T cells, ICOS delivered a potent PI3K signal that was critical for the induction of the key T FH cytokines, IL-21 and IL-4. Under the same settings, CD28 was unable to activate PI3K but supported a robust secondary expansion of T cells. Thus, our results demonstrate a nonredundant function of ICOS-PI3K pathway in the generation of T FH and suggest that CD28 and ICOS play differential roles during a multistep process of T FH differentiation.CD28 ͉ follicular B helper T-cell ͉ germinal center ͉ ICOS ͉ PI3K

LAPTM5 is a lysosomal transmembrane protein expressed in immune cells. We show that LAPTM5 binds the ubiquitin-ligase Nedd4 and GGA3 to promote LAPTM5 sorting from the Golgi to the lysosome, an event that is independent of LAPTM5 ubiquitination. LAPTM5 contains three PY motifs (L/PPxY), which bind Nedd4-WW domains, and a ubiquitin-interacting motif (UIM) motif. The Nedd4–LAPTM5 complex recruits ubiquitinated GGA3, which binds the LAPTM5-UIM; this interaction does not require the GGA3-GAT domain. LAPTM5 mutated in its Nedd4-binding sites (PY motifs) or its UIM is retained in the Golgi, as is LAPTM5 expressed in cells in which Nedd4 or GGA3 is knocked-down with RNAi. However, ubiquitination-impaired LAPTM5 can still traffic to the lysosome, suggesting that Nedd4 binding to LAPTM5, not LAPTM5 ubiquitination, is required for targeting. Interestingly, Nedd4 is also able to ubiquitinate GGA3. These results demonstrate a novel mechanism by which the ubiquitin-ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination.

To identify the structural determinants necessary for opioid receptor desensitization, we serially ablated potential phosphorylation sites in the carboxyl tail of the receptor and examined their effects on [D-Ala 2 ,N-MePhe 4 ,Gly-ol 5 ]enkephalin (DAMGO)-induced desensitization. First, we replaced Thr 394 with alanine (T394A) and stably expressed this mutant receptor in Chinese hamster ovary cells. The T394A receptor did not desensitize after 1 h of treatment with DAMGO, indicating that Thr 394 is required for agonist-induced early desensitization. To test whether Thr 394 was the only residue necessary, we investigated the importance of 7 potential phosphorylation sites between residues 363 and 383, which were all replaced by alanines with the Thr 394 maintained. This mutant (AT) showed partial loss of desensitization (30%), which was attributable to the Ala mutation at Thr 383 , since complete desensitization was achieved by restoring Thr 383 (ATT). These results suggest that Thr 394 is the primary recognition site for G protein-coupled receptor kinases, but Thr 383 is also required for complete agonist-induced desensitization. The specificity of Thr 394 as the primary initiation site appears to be dependent on the preceding acidic amino acid stretch, because in a mutant in which glutamic acid residues at 388, 391, and 393 were replaced by glutamines (EQ), agonist-induced desensitization was completely abolished, identical to the T394A mutant.Desensitization is defined as loss of a biological response to a ligand despite its continuous presence. This phenomenon of desensitization is observed with almost every receptor, and some of the mechanisms involved in desensitization of G protein-coupled receptors have been well characterized using the ␤-adrenergic receptor (␤AR) 1 and rhodopsin. For these receptors, the primary mechanism of desensitization appears to be receptor phosphorylation mediated by G protein-coupled receptor kinases (GRKs). Since these kinases have the unique feature of phosphorylating receptors only when they are in an active or a ligand-bound conformation, GRK-mediated phosphorylation is considered to be the main mechanism for agonist-induced desensitization. These kinases have been shown to phosphorylate a number of G protein-coupled receptors including the ␤AR (1, 2), rhodopsin (3, 4), ␣ 2 -adrenergic receptor (␣ 2 AR) (5, 6), platelet-activating factor receptor (7), thrombin receptor (8), and C5a anaphylatoxin receptor (9).Endogenous opioids play an important role in a variety of physiological processes through pharmacologically distinct, three major receptor subtypes that are coupled to inhibitory G proteins (10). Although the acute action of opioids can induce a number of beneficial effects, chronic use of opioids produces tolerance and dependence (11, 12), which are among the major factors limiting the clinical use of opioids. The molecular mechanisms underlying these phenomena are poorly understood, and receptor desensitization has been implicated as a possible mechanism.It has been sho...

Determining which domains and amino acid residues of the mu opioid receptor are phosphorylated is critical for understanding the mechanism of mu opioid receptor phosphorylation. The role of the C-terminus of the receptor was investigated by examining the C-terminally truncated or point-mutated mu opioid receptors in receptor phosphorylation and desensitization. Both wild-type and mutated receptors were stably expressed in Chinese hamster ovary (CHO) cells. The receptor expression was confirmed by receptor radioligand binding and immunoblottting. After exposure to 5 microM of DAMGO, phosphorylation of the C-terminally truncated receptor and the mutant receptor T394A was reduced to 40 and 10% of that of the wild-type receptor, respectively. Mutation effects on agonist-induced desensitization were studied using adenylyl cyclase inhibition assays. The C-terminally truncated receptor and mutant receptor T394A both showed complete loss of DAMGO-induced desensitization, while the mutant T/S-7A receptor only lost part of its ability to desensitize. Taken together, these results suggest that the C-terminus of the mu opioid receptor participates in receptor phosphorylation and desensitization with threonine 394, a crucial residue for both features. DAMGO-induced mu opioid receptor phosphorylation and desensitization are associated and appear to involve both the mu opioid receptor C-terminus and other domains of the receptor.