Expression and characterization of two alternatively spliced isoforms of rat type I receptor for inositol 1,4,5-trisphosphate

Doris Da May Lin, Yale University.

This is an Open Access Thesis


The transmembrane receptor for IP$\sb3$ is a tetrameric calcium channel located in the endoplasmic reticulum. In many cell types the receptor mediates such complex signals as calcium waves and oscillations. There are multiple receptor isoforms, derived by alternative splicing as well as different genes. The functional significance of these many receptor isoforms has not been determined. To this end, I have generated cell lines that express homotetrameric receptors.Stable lines of the human embryonic kidney (HEK293) cells were established by transfection with cDNAs encoding for two splice variants of the rat type I receptor. The two variants differ in the presence (designated G) or absence (designated F) of a 15-amino acid segment, SI near the ligand-binding domain Both lines expressed a protein of molecular weight $\sim$260 kD, which was identified by sequence-specific polyclonal antibodies. Indirect immunofluorescence carried out on these cells localized the receptor to the endoplasmic reticulum. Like the cerebellar receptor, the expressed protein was a substrate (or cAMP-dependent protein kinase, and contained N-linked sugars. Moreover, the receptor isolated under non-denaturing conditions formed a high-molecular weight complex with a sedimentation profile consistent of a homotetramer.The homotetrameric receptors exhibited complex binding properties. There were multiple states in which the receptor bound IP$\sb3$ cooperatively with different affinities, causing apparent inflections in the binding isotherm. These components may reflect distinct binding sites, or a single class of sites with altered affinities. Calcium acted as an allosteric modulator, shifting the receptor from a high-affinity state (K$\sb{d}\sim $150-20 nM) to a low-affinity state (K$\sb{d}\sim$180-200 nM). The inflections in the IP$\sb3$ binding isotherms remained unaltered in the presence of calcium.While the two receptor isoforms had similar affinities for IP$\sb3$, they interacted with calcium differently. Calcium promoted a sharp decrease of ligand binding in receptor subtype F but a rather gradual decline in G. When receptor function was monitored in live cells by imaging agonist-induced calcium release, the two cell lines also displayed different kinetics of the calcium wave form.