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Lipocalin Receptors: characterisation and structure/function relationships

Reference	BB/D000998/1
Principal Investigator / Supervisor	Professor John B C Findlay
Co-Investigators / Co-Supervisors
Institution	University of Leeds
Department	Inst of Molecular & Cellular Biology
Funding type	Research
Value (£)	241,822
Status	Completed
Type	Research Grant
Start date	01/10/2005
End date	30/09/2008
Duration	36 months

Abstract

Objective (1) The cloning of the first validated receptor for a lipocalin was achieved via the demonstration of an interaction between tear lipocalin (Lcn1) and a phage bearing what turned out to be the N-terminal putative extracellular domain of the intact receptor. Subsequent work confirmed this region as the receptor site for lipocalin binding. Comparison of receptor homologue sequences in the human genome show that they are most similar in the putative transmembrane segments and most variant in the putative water soluble regions, particularly the N-terminal segment and a central potential intracellular loop. The strategy will be to express whole receptors or chimeras of the tear lipocalin receptor possessing the variant N-terminal regions conjoined with the conserved membrane domain. These constructs will then be examined for their ability to bind specifically a selected group of lipocalins using classical binding studies and surface plasmon resonance. Objective (2) (i) It is not known whether the current receptor family members exist naturally as homo- or hetero-oligomers. We will utilise the now well-developed split ubiquitin 2-hybrid system for integral membrane proteins to identify proteins with which the tear lipocalin receptor (LIMR) interacts. This will help define its oligomeric status. These studies will be complemented by gel filtration chromatography of solubilised native and expressed receptors using Western blots with anti-N-terminal antibodies and lipocalin binding as the monitoring procedures. Co-expression of differentially tagged receptors and analysis of the pull-down fraction will also be employed. The study will also begin to reveal the interacting networks in which these receptors participate. (ii) It may be that the functionally best characterised receptor systems, that for retinol binding protein (RBP) may not belong to this cloned family. In which case, RBP based affinity chromatography along with mass spectrometry will be used to identify the receptor. This may also be achieved using a modified split ubiquitin system, along with a placental library. It will be validated by heterologous expression in insect larval cells, by ligand binding studies and by comparison with published data on its functional characteristics. The oligomeric state / complex formation of this new receptor will then be investigated as outlined in objective (2i) above. Objective (3) Using HEK cells which possess both the RBP and the VEG receptors, we will carry out up-take experiments to differentiate between ligand up-take and endocytosis. The specificity of the uptake system will be established by using a relatively non-specific lipocalin (MUP) to which is annealed the 'cd' loop of RBP (in place of its native one) allowing the resultant chimaera (MUPcd) to interact with the RBP receptor. Objective (4) Understanding the structure / function relationships and the main features of an integral membrane protein is greatly facilitated by delineating its topography in the bilayer. This objective is especially significant in this case because of the multiple transmembrane passes suggested by hydropathy analysis. To further this end, we will use the introduction of glycosylation sites and HA epitopes into the putative loop regions and examine the constructs for glycosylation using in vitro expression and for antibody interaction using insect larval cells. The study will be carried out on both the LIMR and the RBP receptor, if from a different family. This will provide a good platform for further mutagenic studies into the molecular basis of the function of these receptors, for example by altering transmembrane segments and intracellular loops that may mediate the uptake of ligand or lipocalin or may provide binding sites for interacting proteins.

Summary

The lipocalins are a family of proteins that have very similar overall conformations but which individually possess unique elements of sequence and structure which largely dictate their biological function. This function is to bind and deliver particular compounds to particular sites where they can exert a profound biological action. Thus vitamin A is bound by retinol binding protein in blood and delivered to peripheral tissues such as the eye where it is converted into a close metabolite which allows the detection of light in vision. Other lipocalins participate in the transmission of pheromones, which influence animal behaviour, in the capture and elimination of odours in the olfactory process, in the delivery of retinoic acid to some developing cell systems, in colouration in invertebrates and plants, in oocyte fertilisation and so on. It has been established in a large number of studies that the mechanism of action of some lipocalins involves binding to cell surface receptors. Recently, unequivocal evidence for the cloning of a membrane receptor for a lipocalin found at a number of sites, including tears, has been obtained. Inspection of the genome indicates that this is only one of a family of at least 7 closely related genes, suggesting that they are each responsible for interacting with specific lipocalins. This project will attempt, therefore, to match up some of these gene products with their interacting lipocalins. It will then go on to define how they associate with the plasma membrane in structural terms and what the molecular composition of the active entity is, eg number and identity of subunits involved. This will in turn allow studies to be conducted into the function of the receptor - how and where they bind their specific lipocalin, what the mechanism of action is, what post-translational modifications, if any, does it undergo and which parts of the receptor are principally involved in these processes. Finally, there may be more than one family ofproteins that act as receptors for lipocalins. The project will, therefore, use so called 2-hybrid techniques in an attempt to identify receptors for those lipocalins which have been shown to possess them but which do not interact with the proteins encoded by any of the genes so far identified by bioinformatic comparisons. The system will also allow other proteins with which the receptor interacts to be identified.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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