A Single Residue in a Novel ADP-ribosyl Cyclase Controls Production of the Calcium-mobilizing Messengers Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate

Latha Ramakrishnan,Hélène Muller-Steffner,Christophe Bosc,Victor D Vacquier,Francis Schuber,Marie-Jo Moutin,Leslie Dale,Sandip Patel

doi:10.1074/jbc.m110.105312

Abstract

Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate are ubiquitous calcium-mobilizing messengers produced by the same family of multifunctional enzymes, the ADP-ribosyl cyclases. Not all ADP-ribosyl cyclases have been identified, and how production of different messengers is achieved is incompletely understood. Here, we report the cloning and characterization of a novel ADP-ribosyl cyclase (SpARC4) from the sea urchin, a key model organism for the study of calcium-signaling pathways. Like several other members of the ADP-ribosyl cyclase superfamily, SpARC4 is a glycoprotein targeted to the plasma membrane via a glycosylphosphatidylinositol anchor. However, unlike most other members, SpARC4 shows a remarkable preference for producing cyclic ADP-ribose over nicotinic acid adenine dinucleotide phosphate. Mutation of a single residue (tyrosine 142) within a noncanonical active site reversed this striking preference. Our data highlight further diversification of this unusual enzyme family, provide mechanistic insight into multifunctionality, and suggest that different ADP-ribosyl cyclases are fine-tuned to produce specific calcium-mobilizing messengers.

Highlights

Phosphate (NAADP) play critical roles in the generation of agonist-evoked calcium signals [2, 3]. cyclic ADP-ribose (cADPR) releases calcium through activation of the ryanodine receptors located on endoplasmic reticulum stores [2, 4]
E-mail: patel.s@ucl.ac.uk. 3 The abbreviations used are: cADPR, cyclic ADP-ribose; NAADP, nicotinic acid adenine dinucleotide phosphate; HEK, human embryonic kidney; PBS, phosphate-buffered saline; RACE, rapid amplification of cDNA ends; GPI, glycosylphosphatidylinositol; HPLC, high pressure liquid chromatography; phosphate (NAADP) play critical roles in the generation of agonist-evoked calcium signals [2, 3]. cADPR releases calcium through activation of the ryanodine receptors located on endoplasmic reticulum stores [2, 4]
These channels have recently been identified as the two-pore channels (10 –12). Both cADPR and NAADP are synthesized by the same family of enzymes, the ADP-ribosyl cyclases [13, 14], which play a central role in calcium signaling and have been implicated in a variety of processes ranging from bacterial clearance [15] to social behavior [16]

Summary

Introduction

Phosphate (NAADP) play critical roles in the generation of agonist-evoked calcium signals [2, 3]. cADPR releases calcium through activation of the ryanodine receptors located on endoplasmic reticulum stores [2, 4]. The mechanism of action of NAADP is less certain [5], in sea urchin eggs, where its effects were discovered [6], it is clear that NAADP acts on novel calcium-permeable channels located not on the endoplasmic reticulum but instead on acidic lysosome-like stores [7,8,9] These channels have recently been identified as the two-pore channels (10 –12). ADP-ribosyl cyclase activity was originally identified in sea urchin egg homogenates [17], but an enzyme with this activity was later purified [18] and cloned [19] from Aplysia californica ovotestes This enzyme was shown to catalyze the cyclization of NAD to form cADPR [18, 20] and the replacement of the nicotinamide moiety in NADP with nicotinic acid to form NAADP [21]. Both SmNACE [24] and SpARC1 [25], able to cyclize nicotinamide guanine dinucleotide to cyclic GDP ribose, produce much less cADPR from NAD [24] than CD38, whereas SpARC2 [55] and several unidentified mammalian ADP-ribosyl cyclases [32, 36, 39] are unable to cyclize nicotinamide guanine dinucleotide

Methods

Results

Conclusion