Role Of Intracellular Domain Research Articles

RIP at the Synapse and the Role of Intracellular Domains in Neurons.

Regulated intramembrane proteolysis (RIP) occurs in a cell when transmembrane proteins are cleaved by intramembrane proteases such as secretases to generate soluble protein fragments in the extracellular environment and the cytosol. In the cytosol, these soluble intracellular domains (ICDs) have local functions near the site of cleavage or in many cases, translocate to the nucleus to modulate gene expression. While the mechanism of RIP is relatively well studied, the fate and function of ICDs for most substrate proteins remain poorly characterized. In neurons, RIP occurs in various subcellular compartments including at the synapse. In this review, we summarize current research on RIP in neurons, focusing specifically on synaptic proteins where the presence and function of the ICDs have been reported. We also briefly discuss activity-driven processing of RIP substrates at the synapse and the cellular machinery that support long-distance transport of ICDs from the synapse to the nucleus. Finally, we describe future challenges in this field of research in the context of understanding the contribution of ICDs in neuronal function.

Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α(2A)-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by G(αq) but not G(α12). The compound inhibited thrombus formation in vivo following vascular injury with an IC(50) of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation.

Role of intracellular domains in the function of the herg potassium channel

The functional role of the large intracellular regions (which include the cyclic nucleotide binding domain, cNBD, and the Per-Arnt-Sim domain, PAS) in the herg channel is not well understood. We have studied possible interactions of the cNBD with other parts of the channel protein using lysine mutations to disrupt such interactions. Some lysine mutations caused significant right shifts in the voltage dependence of inactivation; almost all the mutants caused speeding up of deactivation time course. In a homology model of the cNBD, lysine mutations that affected both inactivation and deactivation lie in a hydrophobic band on the surface of the structure of this domain. Some known mutations in the Long QT Syndrome type 2, with effects on deactivation, are located at residues close to hydrophobic bands on the cNBD and the PAS domains. Such bands of residues in these intracellular domains may play an important part in channel function.

ITIM-dependent endocytosis of CD33-related Siglecs: role of intracellular domain, tyrosine phosphorylation, and the tyrosine phosphatases, Shp1 and Shp2

The leukocyte CD33-related sialic acid-binding Ig-like lectins (Siglecs) are implicated in glycan recognition and host defense against and pathogenicity of sialylated pathogens. Recent studies have shown endocytosis by CD33-related Siglecs, which is implicated in clearance of sialylated antigens and antigen presentation and makes targeted immunotherapy possible. Using CD33 as a paradigm, we have now investigated the reasons underlying the comparatively slow rate of endocytosis of these receptors. We show that endocytosis is largely limited and determined by the intracellular domain while the extracellular and transmembrane domains play a minor role. Tyrosine phosphorylation, most likely through Src family kinases, increases uptake of CD33 depending on the integrity of the two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Simultaneous depletion of the protein tyrosine phosphatases, Src homology-2-containing tyrosine phosphatase 1 (Shp1) and Shp2, which bind to phosphorylated CD33, increases internalization of CD33 slightly in some cell lines, whereas depletion of spleen tyrosine kinase (Syk) has no effect, implying that Shp1 and Shp2 can dephosphorylate the ITIMs or mask binding of the phosphorylated ITIMs to an endocytic adaptor. Our studies show that restraint of CD33 internalization through the intracellular domain is relieved partly when the ITIMs are phosphorylated and show that Shp1 and Shp2 can modulate this process.