Elsevier

Cellular Signalling

Volume 23, Issue 1, January 2011, Pages 213-221
Cellular Signalling

Identification of polycystin-1 and Gα12 binding regions necessary for regulation of apoptosis

https://doi.org/10.1016/j.cellsig.2010.09.005Get rights and content

Abstract

Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations in PKD1, the gene for polycystin-1 (PC1), a transmembrane protein with a cytoplasmic C-terminus that interacts with numerous signaling molecules, including Gα12. The functions of PC1 and the mechanisms of cyst development leading to renal failure are complex. Recently, we reported that PC1 expression levels modulate activity of Gα12-stimulated apoptosis (Yu et al., J. Biol. Chem. 2010 285(14):10243-51). Herein, a mutational analysis of Gα12 and PC1 was undertaken to identify regions required for their interaction and ability to modulate apoptosis. A set of Gα12 mutations with systematic replacement of six amino acids with NAAIRS was tested for binding to the PC1 C-terminus in GST pulldowns. Additionally, a series of deletions within the PC1 C-terminus was examined for binding to Gα12. We identified 3 NAAIRS substitutions in Gα12 that completely abrogated binding, and identified a previously described 74 amino acid Gαi/o binding domain in the PC1 C-terminus as necessary for Gα12 interaction. The functional consequences of uncoupling PC1/Gα12 binding were studied in apoptosis assays utilizing HEK293 cells with inducible PC1 overexpression. Gα12 mutants deficient in PC1 binding were refractory to PC1 inhibition of Gα12-stimulated apoptosis. Likewise, deletion of the Gα12-interacting sequence from the PC1 cytoplasmic domain abrogated its inhibition of Gα12-stimulated apoptosis. Based on the crystal structure of Gα12, the PC1 interaction sites are likely to reside on exposed regions within the G protein helical domain. These structural details should facilitate the design of reagents to uncouple PC1/Gα12 signaling in ADPKD.

Introduction

Autosomal dominant polycystic kidney disease (ADPKD) develops as the result of mutations in the genes PKD1 (~ 70–85%) or PKD2 (~ 15–30%) that encode the protein products polycystin-1 (PC1) and polycystin-2 (PC2) respectively. PC1 is an ~ 460 kDa, eleven transmembrane spanning protein containing an extensive extracellular domain and a relatively short (~ 225 amino acid) cytoplasmic domain that interacts with numerous signaling molecules including trimeric G proteins [1], [2], [3]. PC1 is localized in cilia and at sites of cell–matrix and cell–cell interactions [4], [5], [6]. Mutations in PKD1 lead to defects in cilia function and changes in epithelial cell growth/apoptosis, cell–cell and cell–matrix interactions. Many mutations have been identified in PKD1, and most are deletion, frameshift or nonsense mutations that lead to inactivation of one allele. However, disease development requires an additional insult (a somatic mutation or other injury) to promote cyst development and progression [7], [8]. PKD1 mRNA and protein are expressed throughout nephron development and at moderate to low levels in collecting ducts and distal tubules in the adult. With the development of ADPKD, PC1 protein levels are increased about two-fold [9], [10]. Homozygous loss of PKD1 is embryonic lethal with diffuse cystic disease [11] and conditional knockouts of PKD1 reveal important roles during renal development and have yielded new insights into the mechanisms necessary for cyst formation and progression in vivo (see [12]). Although loss of PC1 leads to cyst development, there is also evidence that PC1 overexpression results in cystic disease [13], [14]. In patients with ADPKD, PC1 expression persists and is even enhanced in most but not all cysts [5], [15]. In addition, transgenic mice overexpressing PC1 develop PKD with renal failure suggesting that, in some cases, a gain of function may be a pathogenic mechanism.

Disregulated apoptosis is an important feature of ADPKD; for instance, increased apoptosis was detected in polycystic kidneys from patients with and without renal failure, but not in controls [16]. Animal models of PKD have also revealed important roles for apoptosis in cyst development in combination with changes in proliferation [17]. However, the focal nature of cyst development, the slow time course of progression, and the changes in apoptosis/proliferation in specific nephron segments at different developmental time points have made identifying the role(s) of apoptosis in disease progression difficult to analyze. We recently demonstrated that PC1 expression levels determine the activity of the Gα12/JNK apoptosis pathway in MDCK cells [18] suggesting a titration mechanism of regulation. Furthermore, we found that Gα12 but not the closely related G protein α-subunit Gα13 bound to the PC1 C-terminus.

In canonical G protein signaling, ligand binding to a G protein coupled receptor (GPCR) results in conformational changes in the Gα subunit that trigger dissociation of GDP and loss of affinity for the Gβγ dimer. GTP rapidly binds to Gα, and signaling through Gα and Gβγ subunits occurs until the intrinsic GTPase activity of Gα hydrolyzes GTP to GDP. G proteins also interact with numerous regulatory and scaffolding proteins. PC1 has been reported to function as an atypical GPCR, bind Gαo/i, and regulate calcium flux through PC2 (a member of the TRP family of calcium channels) by a release of Gβγ subunits [19], [20]. Multiple G protein α-subunits [1], [2], [3] and at least one Regulator of G protein Signaling (RGS) protein [21] interact with PC1. Furthermore, we reported the binding of wildtype and mutationally activated (GTPase deficient) Gα12 to the PC1 C-terminus [3] and recently extended this observation to show that thrombin-stimulated Gα12 preferentially bound to this PC1 domain [18]. Furthermore, in transient overexpression systems, Gα12 regulated AP1 transcription factor activity in a PC1 dependent manner [2]. Herein, we utilize mutant forms of Gα12 and PC1 to provide insights into the structural details of PC1/Gα12 binding, and demonstrate for the first time that apoptotic regulation can be uncoupled by disrupting the interaction between Gα12 and PC1. Based on the Gα12 crystal structure, the PC1 binding sites on Gα12 can be modeled, and implications for regulating the PC1/Gα12 interaction and its effects on apoptosis are discussed.

Section snippets

Chemicals, antibodies and cDNA constructs

Anti-Gα12 (sc-409) and PC1 antibodies (7e12) were purchased from Santa Cruz Biotechnology, and alkaline phosphatase-conjugated anti-rabbit antibody was from Promega. Glutathione-sepharose was from Amersham Pharmacia Biotech. The PC1 C-terminal GST fusion protein was previously established and purified as described [3]. Substitution mutants within myc-tagged QLGα12, in which regions of the cDNA encoding consecutive sextets of amino acids are replaced with a sequence encoding the sextet NAAIRS,

Specific NAAIRS mutations in Gα12 uncouple interaction with PC1 C-terminus

To identify regions of Gα12 important for interaction with PC1, we utilized a series of mutants that systematically replace six amino acids of the Gα12 sequence with the sequence asparagine–alanine–alanine–isoleucine–arginine–serine (NAAIRS). This series of mutations was created within the QLα12 coding sequence to facilitate the study of Gα12 binding proteins that would be predicted as downstream effectors in canonical G protein signaling. We have previously utilized this approach to map the

Discussion

ADPKD is a complex disorder, and the biologic functions of PC1 are only partially understood. PC1 has a large extracellular domain, eleven transmembrane domains, and a short cytoplasmic C-terminus that interacts with numerous signaling molecules. Understanding how this domain regulates intracellular signaling and interactions of PC1 with other proteins is essential to unraveling the cellular physiology of PC1 and the pathology of cyst development and progression. Several lines of evidence

Conclusions

These studies demonstrate that direct binding of PC1 with a G protein α subunit is required for the regulation of a specific signaling pathway.

NAAIRS mutants reveal key structural details modulating the interaction of Gα12 with the PC1 C-terminus.

Mutations of Gα12 in its PC1-binding regions do not affect the ability to stimulate apoptosis yet are uncoupled from PC1 regulation.

The PC1 C-terminal region responsible for binding Gα12 is also essential for regulating apoptosis.

There is growing

Acknowledgments

The authors would like to thank the members of the Harvard Polycystic Kidney Center for their helpful comments and suggestions. This work was supported by the Polycystic Kidney Center P50 DK074030 (JZ and BMD) and GM55223 to BMD; the North Carolina Biotechnology Center (BRG1229) and the NIH (CA100869); and the Lineberger Comprehensive Cancer Center (Affiliate Award, University Cancer Research Fund) to TEM.

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