Solubilization of Receptors for the Novel Ca2+-mobilizing Messenger, Nicotinic Acid Adenine Dinucleotide Phosphate

Georgina Berridge,George Dickinson,John Parrington,Antony Galione,Sandip Patel

doi:10.1074/jbc.m203224200

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+) mobilizing agent in a variety of broken and intact cell preparations. In sea urchin egg homogenates, NAADP releases Ca(2+) independently of inositol trisphosphate or ryanodine receptor activation. Little, however, is known concerning the molecular target for NAADP. Here we report for the first time solubilization of NAADP receptors from sea urchin egg homogenates. Supernatant fractions, prepared following Triton X-100 treatment, bound [(32)P]NAADP with similar affinity and selectivity as membrane preparations. Furthermore, the unusual non-dissociating nature of NAADP binding to its receptor was preserved upon solubilization. NAADP receptors could also be released into supernatant fractions upon detergent treatment of membranes prelabeled with [(32)P]NAADP. Tagged receptors prepared in this way, were readily resolved by native gel electrophoresis as a single protein target. Gel filtration and sucrose density gradient centrifugation analysis indicates that NAADP receptors are substantially smaller than inositol trisphosphate or ryanodine receptors, providing further biochemical evidence that NAADP activates a novel intracellular Ca(2+) release channel.

Highlights

Changes in cytosolic Ca2ϩ are indispensable for normal cell function [1]
Solubilization of nicotinic acid adenine dinucleotide phosphate (NAADP) Receptors—We and others have previously reported binding of [32P]NAADP to membrane preparations derived from sea urchin eggs [26, 28, 29]
In the present study, washed sea urchin egg homogenates were treated with the non-ionic detergent Triton X-100 and supernatants, following ultracentrifugation, analyzed for [32P]NAADP binding

Summary

Introduction

Changes in cytosolic Ca2ϩ are indispensable for normal cell function [1]. A multitude of cell surface Ca2ϩ channels have been characterized that mediate influx of Ca2ϩ from the extracellular space upon activation. Gel filtration and sucrose density gradient centrifugation analysis indicates that NAADP receptors are substantially smaller than inositol trisphosphate or ryanodine receptors, providing further biochemical evidence that NAADP activates a novel intracellular Ca2؉ release channel. Washed sea urchin egg homogenates were solubilized with Triton X-100 (1%) and binding of [32P]NAADP (0.2 nM) to supernatant fractions determined in the presence of the indicated concentrations of unlabeled NAADP (closed circles, n ϭ 8), 2Ј,3Ј-cyclic NAADP (open circles, n ϭ 3) and 3Ј-NAADP (squares, n ϭ 3) added simultaneously.

Results

Conclusion