212 Molecular Brain Research, 17 (1993) 212-216 © 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00 BRESM 70560 of ne Inositol (1,4,5)-trisphosphate receptor: characterization ain and peripheral tissues Michael J. Schell, Sonye K. Danoff and Christopher A. Ross Laboratory of Molecular Neurobiology, Departmentsof Neuroscience and Psychiatry, Johns Hopkins University, Medical School, Baltimore, MD 21205-2196 (USA) (Accepted 30 September 1992) Key words: Second messenger; Inositol lipid; Alternative splicing; Calcium channel; Protein kinase A One ~urce of diversity in the inositol (I,4,5)-trisphosphate receptors (IP3Rs) is generated at the level of alternative splicing. Our previous studies of splice isoforms of the receptor it~ various tissues suggested that some tissues, specifically those containing neurons, selectively express a 40 amino acid insert located between 2 sites for phosphorylation by cyclic AMP-dependent protein kinase (PKA), and that the presence of this insert changes the preferred site of phosphorylation of the receptor. Studies of the mouse receptor have also suggested the existence of intermediately spliced forms containing partial versions of the splice and exhibiting different brain distributions, in this study, we have investigated the alternative splicing of the rat receptor in greater detail using RNase protection and PCR analysis. We find little evidence for the existence of intermediately spliced forms in ra~, raising the possibility that the degree of alternative splicing at this site differs in the brains of two very similar species. Our screen of tissue distribution supports the selectively neuronal expression of the long spliced form, and suggests that regulation of this receptor in neurons may be different than in other tissues. INTRODUCTION The phosphatidylinositol (PI) cycle constitutes an important second messenger system in neurons and other cell types 1'7'9. In this system, neurotransmitter, hormone, or cytokine stimulation of cell surface recep- tors activates a phosphoinositide-specific phospholi- pase C (PLC), generating the intracellular second mes- sengers inositol 1,4,5-trisphosphate (IP 3) and diacyl- glycerol. IP3 binds to an intracellular receptor, the IP3 receptor (IP3R) 6'29, causing the release of calcium from endoplasmic reticulum stores. The IP3R is a tetramer composed of 313 kDa subunits t4aT.Is. Each subunit can bind IP3; the tetramer forms the calcium channel. Expression studies 8,t6.18 indicate that the IP3 binding domain lies near the N-terminus of the protein, while the channel is formed by the transmembrane regions located toward the C-terminus. Between them is a region te~ed the 'coupling domain.' IP3 binding is believed to cause a conformational change transmitted through the coupling domain, opening the channel. The coupling domain may be a site of channel regula- tion, as it contains two cyclic AMP-dependent phos. phorylation sites s,~7. Indeed, phosphorylation of cere. bellar cell membranes by cAMP-dependent protein kinase (PKA) dramatically reduces the potency of IP~ to cause calcium release without changing receptor affinity 2s. Diversity in IP3R structure has been found to occur in at least two ways 15. First, several different genes code for IP3Rs. In the brain, the originally cloned isoform appears to predominate and this is now termed the type I form 27. Two and possibly three other iso- forms are present at lower levels in both brain and peripheral tissues 23.27. A second level of diversity re- sults from alternative splicing of the IP3R. One alter- natively spliced site, termed Sl, lies near the N- terminus; the longer SI spliced form codes for an additional 15 amino acids near the IP3 binding site. SI splice variants show distinct distributions in brain and in peripheral tissues 17a9,2°. Alternative splicing of the IP3R occurs at a second site, located in the coupling domain and termed SII. The long SII splice form codes for an additional 40 Correspondence: C.A. Ross, Laboratory of Molecular Neurobiology, Departments of Neuroscience and Psychiatry, Johns Hopkins University, Medical Sch~l, 720 Rutland Avenue, The Ross Building, Rm. 618, Baltimore, MD 21205-2196, USA. Fax: (1) (410) 614-111113.