Regulation of Inositol 1,4,5-Trisphosphate Receptors by cAMP Independent of cAMP-dependent Protein Kinase

Stephen C Tovey,Skarlatos G Dedos,Taufiq Rahman,Emily J.A Taylor,Evangelia Pantazaka,Colin W Taylor

doi:10.1074/jbc.m109.096016

Abstract

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP3R) to IP3 that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP3R2. Using DT40 cells expressing single subtypes of mammalian IP3R, we demonstrate that high concentrations of cAMP similarly sensitize all IP3R isoforms to IP3 by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP3 binding to IP3R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP3. In single channel recordings from excised nuclear patches of cells expressing IP3R2, cAMP alone had no effect, but it increased the open probability of IP3R2 activated by a submaximal concentration of IP3 alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP3R subtypes to IP3. For IP3R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP3. Using stably expressed short hairpin RNA to reduce expression of the G-protein, Gαs, we demonstrate that attenuation of AC activity by loss of Gαs more substantially reduces sensitization of IP3R by PTH than does comparable direct inhibition of AC. This suggests that Gαs may also specifically associate with each AC·IP3R complex. We conclude that all three subtypes of IP3R are regulated by cAMP independent of PKA. In HEK cells, where IP3R2 selectively associates with AC6, Gαs also associates with the AC·IP3R signaling junction.

Highlights

Ca2ϩ and cAMP are two of a limited number of intracellular messengers used by cells to regulate a diverse array of cellular events in response to many different extracellular stimuli
Potentiation of IP3-evoked Ca2ϩ Release by cAMP—CCh, which activates phospholipase C and IP3 formation via endogenous muscarinic acetylcholine receptors, stimulated Ca2ϩ release from the intracellular stores of HEK-PR1 cells plemented with 10 mM BAPTA
Structure and Function of AC1⁄7IP3R Junctions— We have shown that all three IP3 receptors (IP3R) subtypes are directly regulated by cAMP binding either directly to the IP3R or to a protein tightly associated with it (Figs. 1– 4)

Summary

EXPERIMENTAL PROCEDURES

Cells and Vectors—HEK 293 cells stably transfected with human type 1 PTH receptor (HEK-PR1 cells) were cultured as described [9]. The ORF of rat IP3R1 (GenBankTM accession number GQ233032.1) was amplified by PCR from the expression vector pCMVI-9-. IP3R1 [13] using primers 3 and 4 (supplemental Table S1), and cloned as an EcoRI fragment into pENTR1a vector (Invitrogen). The ORF of mouse IP3R2 (GenBankTM accession number AB182290) was amplified by PCR as two fragments from the expression vector pcDNA3-IP3R2 [14]. The ORF of rat IP3R3 (GenBankTM accession number GQ233031.1) was amplified by PCR from the expression vector pCB6-IP3R3 [15] using primers 8 and 9, and cloned as an EcoRI fragment into pENTR1a vector (Invitrogen). Measurements of IP3-evoked Ca2ϩ Release from Permeabilized Cells—The intracellular stores of DT40 cells stably expressing mammalian IP3R were loaded with Mag-fluo-4 to allow measurement of the luminal-free [Ca2ϩ] [12]. Materials—Sources of materials not specified are provided in a previous study [7]

RESULTS AND DISCUSSION

80 Ϯ 1 82 Ϯ 1

46 Ϯ 12 nM 219 Ϯ 27 nMa

Conclusions