Constitutive Receptor Endocytosis Research Articles

Ligand-selective small molecule modulators of the constitutively active vGPCR US28

US28 is a broad-spectrum constitutively active G protein-coupled receptor encoded by the human cytomegalovirus (HCMV). It binds and scavenges multiple CC-chemokines as well as CX3CL1 (fractalkine) by constitutive receptor endocytosis to escape immune surveillance. We herein report the design and characterization of a novel library of US28-acting commercially available ligands based on the molecular descriptors of two previously reported US28-acting structures. Among these, we identify compounds capable of selectively recognizing CCL2-and CCL4-, but not CX3CL1-induced receptor conformations. Moreover, we find a direct correlation between the binding properties of small molecule ligands to CCL-induced conformations at the wild-type receptor and functional activity at the C-terminal truncated US28Δ300. As US28Δ300 is devoid of arrestin-recruitment and endocytosis, this highlights the potential usefulness of this construct in future drug discovery efforts aimed at specific US28 conformations. The new scaffolds identified herein represent valuable starting points for the generation of novel anti-HCMV therapies targeting the virus-encoded chemokine receptor US28 in a conformational-selective manner.

Identification of a Novel Binding Site Between HIV Type 1 Nef C-Terminal Flexible Loop and AP2 Required for Nef-Mediated CD4 Downregulation

HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.

The Availability of Surface GABAB Receptors Is Independent of γ-Aminobutyric Acid but Controlled by Glutamate in Central Neurons

The Availability of Surface GABAB Receptors Is Independent of γ-Aminobutyric Acid but Controlled by Glutamate in Central Neurons

Role of phospholipase D2 in the agonist‐induced and constitutive endocytosis of G‐protein coupled receptors

We have recently shown that the mu-opioid receptor [MOR1, also termed mu-opioid peptide (MOP) receptor] is associated with the phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane. We further demonstrated that, in human embryonic kidney (HEK) 293 cells co-expressing MOR1 and PLD2, treatment with (D-Ala2, Me Phe4, Glyol5)enkephalin (DAMGO) led to an increase in PLD2 activity and an induction of receptor endocytosis, whereas morphine, which does not induce opioid receptor endocytosis, failed to activate PLD2. In contrast, a C-terminal splice variant of the mu-opioid receptor (MOR1D, also termed MOP(1D)) exhibited robust endocytosis in response to both DAMGO and morphine treatment. We report here that MOR1D also mediates an agonist-independent (constitutive) PLD2-activation facilitating agonist-induced and constitutive receptor endocytosis. Inhibition of PLD2 activity by over-expression of a dominant negative PLD2 (nPLD2) blocked the constitutive PLD2 activation and impaired the endocytosis of MOR1D receptors. Moreover, we provide evidence that the endocytotic trafficking of the delta-opioid receptor [DOR, also termed delta-opioid peptide (DOP) receptor] and cannabinoid receptor isoform 1 (CB1) is also mediated by a PLD2-dependent pathway. These data indicate the generally important role for PLD2 in the regulation of agonist-dependent and agonist-independent G protein-coupled receptor (GPCR) endocytosis.

PKC modulation of GABA A receptor endocytosis and function is inhibited by mutation of a dileucine motif within the receptor β2 subunit

The modulation of GABA A receptors by protein kinase C is complex and involves effects on both ion channel function and receptor trafficking. Although PKC regulates receptor cell surface expression the mechanism is not well understood. Using immunofluorescence studies in HEK 293 cells, we demonstrate that activation of PKC by the phorbol ester PMA promotes receptor endocytosis and is dependent on the presence of a γ subunit. This endocytosis is blocked by the dominant negative dynamin mutant K44A indicating that PKC-induced receptor endocytosis involves the dynamin endocytic pathway. Mutation of a dileucine motif within the receptor β2 subunit inhibits the effect of PKC activation on receptor endocytosis. Using patch clamp analysis, we show that PKC activation produces a robust inhibition of GABA-gated chloride currents in cells expressing wildtype GABA A receptors, but it is ineffective in modulating receptors lacking the dileucine motif. Furthermore, the introduction into the patch pipette of a 10-amino acid peptide corresponding to the dileucine motif present in the receptor β2 subunit prevents PKC modulation of wildtype recombinant receptors. Furthermore, in cerebral cortical neuronal slices inclusion of this peptide in the patch pipette prevents PKC modulation of native GABA A receptors. Using limited chymotrypsin digestion assays, we also show that PKC increases receptor internalization in primary cultures of cerebral cortical neurons. Lastly, PKC inhibitors do not block constitutive receptor endocytosis or affect GABA-gated chloride currents suggesting that PKC-dependent phosphorylation is not required for GABA A receptor endocytosis but plays a modulatory role in the process.

Surface Expression and Endocytosis of the Human Cytomegalovirus-encoded Chemokine Receptor US28 Is Regulated by Agonist-independent Phosphorylation

Human cytomegalovirus encodes the G protein-coupled chemokine receptor homologue US28 that binds several CC chemokines and sequesters extracellular chemokines from the environment of infected cells. Mechanistically, it has been shown that US28 undergoes rapid constitutive receptor endocytosis and recycling. Monoclonal antibodies were raised that allowed the characterization of a ligand-independent phosphorylation and low surface expression of the US28 receptor in transiently transfected HEK293A cells. Phosphoamino acid analysis defined C-terminal serine and threonine residues as phospho-acceptor sites for constitutive receptor phosphorylation. Coexpression of G protein-coupled receptor kinase-2 and US28 enhanced ligand-independent receptor phosphorylation. C-terminal serine to alanine mutagenesis of US28 resulted in a decreased phosphorylation rate that correlated with enhanced surface expression. Maximal surface expression was detected when all C-terminal serines were substituted. Exchange of all C-terminal serines also significantly reduced receptor endocytosis. Thus, constitutive US28 phosphorylation regulates receptor endocytosis and receptor surface display and may thereby provide a pathogenic mechanism for a potential decoy function of the virally encoded receptor.

Role of signal transduction in internalization of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein.

For G protein-coupled receptors, limited information is available on the role of agonist binding or of the second-messenger products of receptor signaling on receptor endocytosis. We explored this problem using the opossum PTH/PTH-related protein (PTHrP) receptor, a prototypical Class II G protein-coupled receptor, as a model. In one approach, we evaluated the endocytic properties of mutated forms of the opossum PTH/PTHrP receptor that we had previously shown to be impaired in their ability to initiate agonist-induced signaling when expressed in COS-7 cells. A point mutation in the third cytoplasmic loop (K382A) that severely impairs PTH/PTHrP receptor signaling significantly reduced internalization, whereas two mutant receptors that displayed only partial defects in signaling were internalized normally. To explore more directly the role of second-messenger pathways, we used a cleavable biotinylation method to assess endocytosis of the wild-type receptor stably expressed in human embryonic kidney (HEK) 293 cells. A low rate of constitutive internalization was detected (<5% over a 30-min incubation at 37 C); the rate of receptor internalization was enhanced about 10-fold by the receptor agonists PTH(1-34) or PTHrP(1-34), whereas the receptor antagonist PTH(7-34) had no effect. Forskolin treatment produced a minimal increase in constitutive receptor endocytosis, and the protein kinase (PK)-A inhibitor H-89 failed to block agonist-stimulated endocytosis. Similarly, activation of PK-C, by treatment with phorbol 12-myristate 13-acetate, elicited only a minimal increase in constitutive receptor endocytosis; and blockade of the PK-C pathway, by treatment with a bisindolylmaleimide, failed to inhibit agonist-induced receptor endocytosis. Immunofluorescence confocal microscopic studies of PTH/PTHrP receptor internalization confirmed the results using receptor biotinylation. These findings suggest that: 1) agonist binding is required for the efficient endocytosis of the PTH/PTHrP receptor; 2) receptor activation (agonist-induced receptor conformational change) and/or coupling to G proteins plays a critical role in receptor internalization; and 3) activation of PK-A and PK-C is neither necessary nor sufficient for agonist-stimulated receptor internalization.