Abstract
Given the interaction of the inositol 1,4,5-trisphosphate receptor (IP(3)R) with chromogranins A (CGA) and B (CGB), two major Ca(2+) storage proteins of secretory granules that have been shown to be IP(3)-sensitive intracellular Ca(2+) store of neuroendocrine cells, we have investigated the potential interaction of the intraluminal loop regions of the IP(3)R with both intact CGB and the conserved near N-terminal region of CGB. The interaction studies carried out with CGB and glutathione S-transferase fusion proteins of intraluminal loop regions of bovine type 1 IP(3)R showed that CGB interacts with intraluminal loop 3-2 (the second loop formed between transmembrane regions 5 and 6) of the IP(3)R at both pH 5.5 and 7.5. Analytical ultracentrifugation studies also indicated that CGB interacts with the same intraluminal loop region of the IP(3)R and the interaction was much stronger than that between CGA and the loop. Moreover, the conserved near N-terminal region of CGB also interacted with the intraluminal loop region of the IP(3)R. The CGB interaction with the IP(3)R intraluminal loop peptide at pH 7.5 showed a DeltaG(0) value of -8.1 kcal/mol at 37 degrees C for a 1:1 stoichiometry, indicating a K(d) of approximately 1.9 micrometer. These results give insight into the molecular organization of the IP(3)-sensitive Ca(2+) store.
Highlights
Since the initial finding that the secretory vesicles of the bovine adrenal medullary chromaffin cells release Ca2ϩ in response to inositol 1,4,5-trisphosphate (IP3)1 [1], the IP3-sensitive intracellular Ca2ϩ store role of the secretory granules has been demonstrated in the zymogen granules of pancreatic acinar cells [2] and in the secretory granules of goblet cells [3]
Unlike the interaction that has been demonstrated between L3-2 and CGB (Fig. 2), the interaction study between the Glutathione S-Transferase (GST) fusion proteins of the intraluminal loop regions of IP3R1 and CGA failed to show interaction between the L3-2 region and CGA
This observation has been extended to the secretory granules of goblet cells that direct participation of secretory granule calcium in the control of cytoplasmic Ca2ϩ concentration has been shown [3]; the uptake of Ca2ϩ by the secretory granules of goblet cells was temporally and spatially matched by simultaneous reduction of Ca2ϩ concentration in the surrounding cytoplasm, whereas IP3-mediated release of Ca2ϩ by the secretory granules resulted in the simultaneous increase of cytoplasmic Ca2ϩ concentration in the immediate vicinity of the secretory granules, clearly demonstrating the participation of secretory granule calcium in the control of cytoplasmic Ca2ϩ concentration
Summary
Vol 275, No 39, Issue of September 29, pp. 30293–30300, 2000 Printed in U.S.A. Interaction of Chromogranin B and the Near N-terminal Region of Chromogranin B with an Intraluminal Loop Peptide of the Inositol 1,4,5-Trisphosphate Receptor*. Given the interaction of the inositol 1,4,5-trisphosphate receptor (IP3R) with chromogranins A (CGA) and B (CGB), two major Ca2؉ storage proteins of secretory granules that have been shown to be IP3-sensitive intracellular Ca2؉ store of neuroendocrine cells, we have investigated the potential interaction of the intraluminal loop regions of the IP3R with both intact CGB and the conserved near N-terminal region of CGB. The CGB interaction with the IP3R intraluminal loop peptide at pH 7.5 showed a ⌬G0 value of ؊8.1 kcal/ mol at 37 °C for a 1:1 stoichiometry, indicating a Kd of ϳ1.9 M These results give insight into the molecular organization of the IP3-sensitive Ca2؉ store. The conserved near N-terminal region of CGA was shown to interact with an intraluminal loop region of the IP3R [29], probably revealing the interacting domains of two proteins. We have investigated the potential interaction of the intraluminal loop region of the IP3R with both intact CGB and the conserved near N-terminal CGB region in the present study and found that intact CGB as well as the conserved near N-terminal region of CGB interact with the intraluminal loop region of the IP3R
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