Localization of Three Types of the Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channel in the Secretory Granules and Coupling with the Ca2+ Storage Proteins Chromogranins A and B

Seung Hyun Yoo,Young Soo Oh,Moon Kyung Kang,Yang Hoon Huh,Seung Ho So,Hyung Seon Park,Hee Yun Park

doi:10.1074/jbc.m107532200

Abstract

Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular Ca(2+) store and the presence of the IP(3) receptor (IP(3)R)/Ca(2+) channel on the secretory granule membrane have been established, the identity of the IP(3)R types present in the secretory granules is not known. We have therefore investigated the presence of different types of IP(3)R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy and found the existence of all three types of IP(3)R in the secretory granules. To determine whether these IP(3)Rs interact with CGA and CGB, each IP(3)R isoform was co-transfected with CGA or CGB into NIH3T3 or COS-7 cells, and the expressed IP(3)R isoform and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP(3)R form complexes with CGA and CGB in the cells. To further confirm whether the IP(3)R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP(3)R and CGA and CGB was tested by co-immunoprecipitation experiments of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP(3)R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP(3)R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca(2+) binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca(2+)/mol with a dissociation constant (K(d)) of 1.5 mm at pH 5.5 but virtually no Ca(2+) at pH 7.5. The high capacity, low affinity Ca(2+)-binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca(2+) storage protein in the secretory granules.

Highlights

The secretory granules of endocrine cells, neurons, and neuroendocrine cells contain many hormones, ions, peptides and proteins, including 40 mM Ca2ϩ and 1–2 mM chromogranins A and B in addition to high concentrations of hormones [1,2,3,4,5,6]
In bovine adrenal medullary chromaffin cells the secretory granules occupy ϳ10% of the total cell volume [8], thereby storing a majority of the intracellular Ca2ϩ of the cell in the secretory granules. It appears inevitable for the secretory granules to participate in the control of intracellular Ca2ϩ concentrations. Consistent with this notion, the secretory granules from adrenal medullary chromaffin cells [9], pancreatic acinar cells [10], and the goblet cells [11] were shown to release stored Ca2ϩ in response to IP3.1 Using optical sectioning and fluorescent microscope techniques the participation of granular Ca2ϩ in the control of intracellular Ca2ϩ concentration has clearly been demonstrated by measuring the Ca2ϩ concentrations of both the intragranular milieu and of the cytoplasm that is immediately adjacent to the secretory granules simultaneously [11]
Visualization of the purified proteins on an SDS-polyacrylamide gel showed proteins with molecular sizes of 260 –280 kDa (Fig. 1A); the proteins purified by type 1 and type 3 specific antibodies were shown in a ϳ280-kDa region while the protein purified by the type 2-specific antibody was shown in a ϳ260-kDa region, indicating that each IP3 receptor (IP3R) isoform-specific antibody interacted with different proteins in the

Summary

Introduction

The secretory granules of endocrine cells, neurons, and neuroendocrine cells contain many hormones, ions, peptides and proteins, including 40 mM Ca2ϩ and 1–2 mM chromogranins A and B in addition to high concentrations of hormones [1,2,3,4,5,6]. The high capacity, low affinity Ca2ϩ binding property of CGA has been proposed to be responsible for the IP3-sensitive Ca2ϩ store role of secretory granules of bovine adrenal medullary chromaffin cells [9, 37].

Results

Conclusion