Systems Biology of the Vasopressin V2 Receptor: New Tools for Discovery of Molecular Actions of a GPCR

Abstract

Systems biology can be defined as the study of a biological process in which all of the relevant components are investigated together in parallel to discover the mechanism. Although the approach is not new, it has come to the forefront as a result of genome sequencing projects completed in the first few years of the current century. It has elements of large-scale data acquisition (chiefly next-generation sequencing-based methods and protein mass spectrometry) and large-scale data analysis (big data integration and Bayesian modeling). Here we discuss these methodologies and show how they can be applied to understand the downstream effects of GPCR signaling, specifically looking at how the neurohypophyseal peptide hormone vasopressin, working through the V2 receptor and PKA activation, regulates the water channel aquaporin-2. The emerging picture provides a detailedframework for understanding the molecular mechanisms involved in water balance disorders, pointing the way to improved treatment of both polyuric disorders and water-retention disorders causing dilutional hyponatremia.

Keywords: diabetes insipidus; hyponatremia; kidney; next-generation DNA sequencing; protein mass spectrometry; syndrome of inappropriate antidiuresis.

Figure 1.. Regulation of water excretion by vasopressin.

(A) Feedback regulation of water excretion and thirst. Plasma osmolality is sensed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus, regulating the secretion of arginine vasopressin (AVP) by the neurohypophysis (posterior pituitary) with concomitant regulation of thirst. The resulting increase in plasma AVP triggers actions in the kidney to increase water reabsorption from the collecting duct pro-urine to the blood, thereby lowering plasma osmolality. (B) AVP regulates water excretion by controlling osmotic water permeability in collecting duct principal cells. AVP binds to the vasopressin V2 receptor, which increases cyclic AMP production by activation of adenylyl cyclase 6 (Adcy6) via the heterotrimeric G-protein alpha subunit G_s. Elevation of cyclic AMP activates protein kinase A (PKA) that triggers increases in trafficking of the water channel aquaporin-2 (Aqp2) to the plasma membrane as well as increases in the total abundance of the Aqp2 protein. Identification of the ‘Unknown Processes’ that link PKA to regulation of Aqp2 is the main topic of this review. (C) A cluster of four phosphorylation sites in the carboxyl terminal tail of Aqp2 are regulated by vasopressin. Serines in positions 256, 261, 264, and 269 (shaded) were identified by Hoffert et al. (29). Immunoblotting with phospho-specific antibodies to each of the four sites showed that each is regulated by vasopressin although with differing time courses (30) (below).

Figure 2.. RNA-seq analysis of vasopressin signaling in mouse collecting duct cells (mpkCCD).

(A) A typical RNA-seq experiment starts with the selection of polyadenylated RNA transcripts using oligonucleotide (dT) primers (Poly-A selection) or depletion of high abundance ribosomal RNA (Ribo-depletion). The resulting RNA is then reverse transcribed to produce cDNA fragments, depending on the RNA-seq methods. Sequencing adapters (red and green bars) are then attached to the cDNA fragments to generate sequencing libraries through PCR amplification. The barcode libraries are deep sequenced using a high-throughput sequencing platform (e.g., Illumina) to obtain short sequencing reads. Sequencing reads, including exon and junction reads (reads with dashed lines), are subsequently aligned to the mouse reference genome to quantify gene expression. (B) Volcano plot for RNA-seq data for all 8393 detectable transcripts in dDAVP and vehicle-treated cells at 24 hrs. A relatively small number of transcripts, including Aqp2, were altered in abundance by vasopressin. The horizontal axis shows the mean log₂(dDAVP/Vehicle) for 9 pairs of samples. The vertical axis shows −log₁₀P for t-tests for each gene. Vertical dashed lines show 95% confidence interval for random variation based on Vehicle:Vehicle comparisons (2 × SD). Panel B was modified from Sandoval et al. (68).

Figure 3.. Small sample RNA-seq in microdissected renal tubules.

(A) Nomenclature for all 14 renal tubule segments. The scheme shows the connection of both a short-looped and a long-looped nephron to the collecting duct system. Definitions: PT-S1, the initial segment of the proximal tubule; PT-S2, proximal straight tubule in cortical medullary rays; PT-S3, last segment of the proximal straight tubule in the outer stripe of outer medulla; DTL1, the short descending limb of the loop of Henle; DTL2, long descending limb of the loop of Henle in the outer medulla; DTL3, long descending limb of the loop of Henle in the inner medulla; ATL, thin ascending limb of the loop of Henle; MTAL, medullary thick ascending limb of the loop of Henle; CTAL, cortical thick ascending limb of the loop of Henle; MD, macula densa; DCT, distal convoluted tubule; CNT, connecting tubule; CCD, cortical collecting duct; OMCD, outer medullary collecting duct; IMCD, inner medullary collecting duct. (B) Distributions of transcript abundances for collecting duct specific genes. TPM, transcripts per million. Panel A was redrawn from Chen et al. (80).

Figure 4.. ChIP-seq.

(A) ChIP-seq procedure. (B) ChIP-seq using an antibody to RNA Polymerase II (Polr2a) reports occupancy of RNA Polymerase II complex over Aqp2 gene in presence and absence of V2 receptor selective vasopressin analog dDAVP. (C) Plot of transcript abundance ratio (dDAVP/Vehicle) versus RNA Polymerase II occupancy ratio (dDAVP/Vehicle) shows that only a few genes are regulated transcriptionally in response to V2 vasopressin receptor binding.

Figure 5.. Enhancer identification.

(A) Protocol for ATAC-seq identification of regions of open chromatin. (B) Mapping of open chromatin regions within the CTCF-loop surrounding the Aqp2 gene. Several potential transcription factors binding motifs were identified in a putative enhancer just 3’ to the Aqp2 gene. Candidate transcription factors are listed.