Subcellular distribution of GABA(B) receptor homo- and hetero-dimers.

GBRs (GABA(B) receptors; where GABA stands for gamma-aminobutyric acid) are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. In vitro assays have previously demonstrated that these receptors are heterodimers assembled from two homologous subunits, GBR1 and GBR2, neither of which is capable of producing functional GBR on their own. We have used co-immunoprecipitation in combination with bioluminescence and fluorescence resonance energy transfer approaches in living cells to assess directly the interaction between GBR subunits and determine their subcellular localization. The results show that, in addition to forming heterodimers, GBR1 and GBR2 can associate as stable homodimers. Confocal microscopy indicates that, while GBR1/GBR1 homodimers are retained in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartment, both GBR2/GBR2 homodimers and GBR1/GBR2 heterodimers are present at the plasma membrane. Although these observations shed new light on the assembly of GBR complexes, they raise questions about the potential functional roles of GBR1 and GBR2 homodimers.

Target validation of G-protein coupled receptors.

G-protein coupled receptors (GPCRs) represent possibly the most important target class of proteins for drug discovery. Over 30% of clinically marketed drugs are active at this receptor family. These drugs exhibit their activity at <10% of all known GPCRs. A major challenge for the pharmaceutical industry is to associate the many novel GPCRs with disease to identify the drugs of the future. This process consists of a collection of experimental paradigms that together can be loosely labelled 'target validation'.

In vitro selection of RNA aptamers that block CCL1 chemokine function.

CCL1, the CCR8 ligand, is a CC chemokine secreted by activated monocytes and lymphocytes and is a potent chemoattractant for these cell types. The in vivo role of the CCL1/CCR8 axis in Th2-mediated inflammation is far from clear. Ligand neutralisation studies reported discrepancies in the effect of CCL1/CCR8 and CCR8 knockout studies showed very different insights into the functional role of the CCR8. To further study the biological function of CCL1, we focused on the generation and characterisation of RNA aptamers. We report here the in vitro isolation of the first nuclease resistant and selective RNA aptamer (T48) with high-binding affinity for human and mouse CCL1. The T48 aptamer but not a random control aptamer antagonises CCL1 function in a dose-dependent fashion in both heparin binding and chemotaxis assays. To our knowledge, the T48 aptamer constitutes one of the most potent CCL1 antagonists reported to date and is an excellent tool to dissect CCL1-specific function in vivo. The T48 aptamer may also have potential as new generation of therapeutic tools.

Characterization of 16 human G protein-coupled receptors expressed in baculovirus-infected insect cells.

Understanding the three-dimensional structure of G protein-coupled receptors (GPCRs) has been limited by the technical challenges associated with expression, purification, and crystallization of membrane proteins, and their low abundance in native tissue. In the first large-scale comparative study of GPCR protein production using recombinant baculovirus, we report the characterization of 16 human receptors. The GPCRs were produced in three insect cell lines and functional protein levels monitored over 72 h using radioligand binding assays. Different GPCRs exhibited widely different expression levels, ranging from less than 1 pmol receptor/mg protein to more than 250 pmol/mg. No single set of conditions was suitable for all GPCRs, and large differences were seen for the expression of individual GPCRs in different cell lines. Closely related GPCRs did not share similar expression profiles; however, high expression (greater than 20 pmol/mg) was achieved for over half the GPCRs in our study. Overall, the levels of protein production compared favourably to other published systems.

Identification of potent and selective RNA antagonists of the IFN-gamma-inducible CXCL10 chemokine.

CXCL10 (also known as IP-10 in humans and CRG-2 in mice) is a nonglycosylated chemokine and a member of the non-ELR CXC chemokine subfamily implicated in a variety of inflammatory conditions. The role of CXCL10 in different disease states still requires clarification, and new approaches are necessary to better understand its biological function. We report here the isolation of a series of nuclease-resistant RNA aptamers that act to antagonize human CXCL10 function in a number of in vitro and cell-based assays. The two most potent aptamers identified were highly selective for human CXCL10. A further aptamer was identified that antagonized both the human and the mouse CXCL10. A combination of a molecular-biology-based truncation and solid-phase synthesis enabled the truncation of one of the aptamers from 71 to 34 nucleotides. This was followed by PEGylation, 3' capping, and further stabilization of the RNA aptamer, while its high potency was maintained. These aptamers could be utilized as powerful target validation tools and may also have therapeutic potential. To our knowledge, the CXCL10 aptamers generated are the most potent antagonists of CXCL10/CXCR3 signaling reported to date.