Developing new methods to study chemokine receptor binding

Samantha Jane Allen, UC San Diego

Membrane proteins constitute approximately 1/3 of all mammalian proteins, and, of these, Gprotein coupled receptors (GPCRs) represent the largest superfamily, with over 800 members in humans. The chemical diversity of their endogenous ligands, and the range of processes that they control are exceptional, ranging from sensation of stimuli such as light, taste and odor, to neurotransmission and cell migration. It is not surprising, therefore, that about 60% of drugs currently available are targeted directly or indirectly at GPCRs.

Despite their importance and abundance, the structure of only one GPCR (rhodopsin) has been solved to high resolution to date. This is mainly due to the degree of difficulty in expressing, functionally reconstituting and crystallising membrane proteins compared to soluble ones. However, if we are to understand more about these proteins and treat diseases associated with them, it is important that we develop methods to study them.

Chemokine receptors, the focus of this study, are GPCRs that control cell migration in the immune response and inflammation, and in developmental processes such as haematopoietic cell and cerebellar development. However, viruses, such as HIV utilise a number of these chemokine receptors, including CCR5, CXCR4 and D6, as essential co-factors during viral entry into host cells. Therefore, these proteins are particularly important to study with respect to the treatment of HIV.

The specific aims of this project are as follows:

  1. To express the human G-protein-coupled chemokine receptors CCR1, D6 and CCR5, concentrating upon a new inducible mammalian expression system that has been shown to facilitate the expression of exceptionally high levels of rhodopsin.
  2. To develop methods to purify and solubilise functional chemokine receptors in artificial detergent/lipid membranes.
  3. To develop high throughput assays to verify the presence of native receptors by the ability to bind labeled chemokines and small molecules.
  4. To study the structure of one or more receptors, using a combination of 19F NMR spectroscopy and x-ray crystallography, in order to understand more about small-molecule binding sites. These studies should aid the development of small molecule-antagonists to treat diseases such as HIV.

The ultimate aim of this study is to use the structural information obtained from these studies to aid the development of more potent and specific therapeutic compounds for treating HIV and other chemokine-receptor related diseases. These studies will be facilitated through an ongoing collaboration with Berlex Biosciences, a company based in Northern California with a strong interest in diseases linked to the chemokine receptors CCR1 and CCR5. Although I am focusing specifically on chemokine receptors and HIV, the results from this study will also provide general information relevant to many other GPCR-related diseases.