Adiponectin identified as an agonist for PAQR3/RKTG using a yeast-based assay system

Abstract

The PAQR family of proteins comprises an intriguing group of newly discovered receptors. Although the agonist is known for 5 of the 11 human PAQRs, most are considered "orphan" receptors. We developed a yeast-based assay system for PAQR receptor activity that can be used to identify agonists for PAQRs of unknown function. Using this system, we found that the proteinaceous hormone adiponectin functions as an agonist of PAQR3, a previously uncharacterized member of this family. This is not surprising given that PAQR3 is most closely related to PAQR1 (AdipoR1) and PAQR2 (AdipoR2), which also sense adiponectin. The identification of adiponectin as an agonist for PAQR3 is of considerable clinical relevance because adiponectin suppresses the proliferation of tumor cells and it has been reported that PAQR3 suppresses tumorigenesis. Thus, the interaction between PAQR3 and adiponectin may help explain the antiproliferative properties of adiponectin.

Figure 1

Functional expression of human PAQRs in yeast. All cells are wild type and are grown in LIM-Fe. (A) Medium contains 0.05% galactose to modestly overexpress GAL1-driven genes. Black squares show FET3-lacZ activity in yeast carrying empty expression vector. This strain carries the genomic copy of IZH2. White squares show activity in yeast carrying a GAL-driven Izh2p overexpression vector in addition to the genomic copy of IZH2. Activity is presented as a percentage of activity measured in a strain that has not been treated with thaumatin. (B) Medium contains 2% galactose to fully induce all human PAQRs. FET3-lacZ activity is presented as a percentage of activity seen in a strain carrying empty expression vector. (C) Medium contains 2% galactose to fully overexpress AdipoR2, mPRα, mPRδ, andmPRε. Cells are exposed to their respective agonists (100 pM adiponectin for AdipoR2 or 10 μM progesterone for mPRδ, mPRα, and mPRε). FET3-lacZ activity is presented as a percentage of activity seen in an untreated strain carrying empty expression vector.

Figure 2

PAQR3 responds to adiponectin. All cells are wild type and grown in LIM-Fe. (A) Medium contains either 2% galactose for full expression (AdipoR2, mPRδ, mPRα, mPRε, and MMD2) or 0.05% galactose for reduced expression (AdipoR1, PAQR3, PAQR4, mPRγ, mPRβ, and MMD1). The effect of adiponectin on FET3-lacZ in cells carrying each PAQR is presented as a percentage of activity seen in untreated cells. (B) Medium contains 0.05% galactose for reduced PAQR expression. Response of each human class I PAQR to adiponectin is shown. For each expressed PAQR, FET3-lacZ activity is presented as a percentage of activity seen in untreated cells carrying empty expression vector.

Figure 3

PAQR3 expression levels. (A) Western blots of total membrane preparations from cells carrying empty expression vector (pGREG536) or HA-tagged AdipoR1 and PAQR3 expression vectors. Blots are probed with an anti-HA antibody. Equal amount of total protein were loaded in each lane. (B) Western blots of the same samples from (A) probed with an antiporin antibody to control for loading of membrane protein extracts. (C) Binding of FAM-labeled adiponectin to spheroplasts of wild-type cells overexpressing AdipoR1. Binding is first determined as a percent of cells with FAM-adiponectin bound for cells carrying either empty expression vector or AdipoR1 expression vector. Data are plotted as percent bound to AdipoR1-expressing cells percent bound to cells—carrying empty vector. Error bars indicate ±1 SD.

Figure 4

Alignment of the third conserved motif in adiponectin receptor homologs. Black shading indicates amino acids that are highly conserved throughout the entire PAQR family. Gray shading indicates amino acids that can be considered diagnostic of AdipoR1/2-like, PAQR3-like, or PAQR4-like proteins. The full names and taxonomic grouping of the organisms from which these sequences are derived are shown in Supplemental Figure 1.