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Title: Characterization of types 1 and 3 inositol trisphosphate receptors
Author: Cardy, T. A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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Rat inositol 1,4,5-trisphosphate (IP3) receptors were expressed using a baculovirus system. Recombinant cDNAs encoding the types 1, 2 and 3 IP3 receptors were used to infect Spodoptera freugiperda (Sf9)cells and membrane proteins were harvested 40 hours later into a cytosol-like medium (CLM). Expressed IP3 receptors were identified using antipeptide antisera specific to the C-terminus of each receptor subtype or to a sequence common to the N-termini of all IP3 receptors. The expressed IP3 receptors were full length (260kDa), types 1 and 3 receptors were expressed at levels 30-40-fold higher than endogenous IP3 receptors, appropriately glycosylated and assembled into homotetramers. In Ca2+-free CLM, type 3 receptors bound [3H] IP3 with 5-fold higher affinity than type 1 receptors. Binding of [3H] IP3 to each subtype was regulated be increases in [Ca2+], but by different mechanisms. An increase in [Ca2+] reversibly inhibited [3H] IP3 binding to type 1 receptors ˜50% by decreasing the number of IP3-binding sites (Bmax) without affecting their affinity for IP3. The effects of Ca2+ on [3H] IP3 binding to type 3 receptors were more complex. Elevating [Ca2+] first stimulated [3H] IP3 binding by increasing Bmax, further increasing [Ca2+] then inhibited binding by decreasing the affinity for IP3. As the [Ca2+] increased therefore, the relative affinities of the type 1 and 3 receptors for IP3 reversed. Radioligand binding analyses with a range of unrelated ligands indicated that structures analogous to the vicinal 4,5-bisphosphate and equatorial 6-hydroxyl of IP3 were essential for high affinity binding to both receptor subtypes. In addition, both the affinity and selectivity for the subtypes were influenced by the 1-phosphate and axial 2-hydroxyl. Functional analyses were impossible with Sf9 cells, I therefore used cell lines (SH-SY5Y for type 1 and RinM5F for type 3) in which I had confirmed the relative levels of expression of each receptor subtype. These functional analyses were consistent with the subtype-selectivity of the ligands identified in binding studies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available