The analysis of heterotaxy patients reveals new loss-of-function variants of GRK5

Abstract

G protein-coupled receptor kinase 5 (GRK5) is a regulator of cardiac performance and a potential therapeutic target in heart failure in the adult. Additionally, we have previously classified GRK5 as a determinant of left-right asymmetry and proper heart development using zebrafish. We thus aimed to identify GRK5 variants of functional significance by analysing 187 individuals with laterality defects (heterotaxy) that were associated with a congenital heart defect (CHD). Using Sanger sequencing we identified two moderately frequent variants in GRK5 with minor allele frequencies <10%, and seven very rare polymorphisms with minor allele frequencies <1%, two of which are novel variants. Given their evolutionarily conserved position in zebrafish, in-depth functional characterisation of four variants (p.Q41L, p.G298S, p.R304C and p.T425M) was performed. We tested the effects of these variants on normal subcellular localisation and the ability to desensitise receptor signalling as well as their ability to correct the left-right asymmetry defect upon Grk5l knockdown in zebrafish. While p.Q41L, p.R304C and p.T425M responded normally in the first two aspects, neither p.Q41L nor p.R304C were capable of rescuing the lateralisation phenotype. The fourth variant, p.G298S was identified as a complete loss-of-function variant in all assays and provides insight into the functions of GRK5.

Figure 1. Variants of human GRK5 identified in heterotaxy patients.

(a) Cartoon displays the 16 exons and the detected genetic variations as well as their respective location within the coding region. The variants are shown at the nucleotide levels (e.g. c.122A < T) as well as the subsequent amino acid change in the protein (e.g. p.Q41L). Red bars mark variants more closely characterised in this study. The p.T425M variant of GRK5 identified in human patients corresponds to the p.S425M variant of zebrafish Grk5l used in this work. (b) Numbers and red spheres indicate location of relevant variants in the structure of bovine GRK5 (PDB entry 4WNK). Only position 298 is absolutely conserved as glycine in GRKs. Image provided by John J. G. Tesmer, University of Michigan, Ann Arbor.

Figure 2. Mutations in Grk5l alter its ability for receptor desensitisation.

We tested the ability of Grk5l variants to terminate signalling of four different receptors in a luciferase reporter assay that is based on the Gq-Rho-SRE signal transduction axis. Individual receptors were transiently expressed in HEK293 cells together with wild-type Grk5l or different variants thereof. As positive control for receptor desensitisation, bovine GRK5 was co-expressed with the receptor. All panels show representative blots. One-way ANOVA was applied and all variants were compared to wild-type Grk5l; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. Bar graphs display mean ± SEM of three independent experiments done in triplicates. (a) Murine AT1a receptor stimulated with 30 μM angiotensin II: All variants except for p.G298S display the same efficiency in the desensitisation of AT1a receptors. (b) Human CCR2b receptor stimulated with 50 nM CCL2: The p.G289S shows significantly reduced ability to terminate signalling compared to wild-type Grk5l, while p.S425M is even more potent. (c) Human CCR3 receptor stimulated with 50 nM CCL11: As CCR3 is not G_q-coupled a Gα_qi5 construct was co-transfected, which enables G_i-receptors to stimulate G_q signalling cascades. Grk5l inhibits signalling to a similar extent as bovine GRK5. The p.G298S variant lacks the ability to desensitise CCR3 receptors, while the lower inhibition seen by p.R304C may be accounted to lower expression levels. (d) Human CXCR4 stimulated with 50 nM CXCL12: As CXCR4 is not G_q-coupled a Gα_qi5 construct was co-transfected, which enables G_i-receptors to stimulate G_q signalling cascades. On its own, Gα_qi5 does not alter receptor signalling. p.G298S and also p.R304C are less effective in inhibiting receptor signalling, whereas p.Q41L and p.S425M appeared to be more potent than wild-type Grk5l.

Figure 3. Subcellular distribution of Grk5l variants.

Upper panel: HEK293 cells were transiently transfected with GFP fusion constructs of wild-type or mutated Grk5l (cloned into pEGFP-N3) and analysed using confocal microscopy. Representative images of three transfections shown. Lower panel: Expression of Grk5l variants in zebrafish produced subcellular distribution of the kinases similar to the results in HEK293 cells. Images show expression in the epidermis. At least five different embryos each from two different injection days were assessed.

Figure 4. Variants of Grk5l localise to cilia.

Human forearm fibroblast cells immortalised by hTERT were transfected with pEGFP-N3 plasmids carrying HA-tagged wild-type or mutated Grk5l cDNA, followed by serum starvation. Cells were then probed for acetylated-tubulin and the HA tag, respectively, to detect primary cilia and expressed Grk5l variants. Assessment by confocal microscopy revealed ciliary localisation of all variants. Acetylated-tubulin is shown in red in panels to the left. Localisation of Grk5l variants are represented in green in middle panels, while panels to the right show an overlay.

Figure 5. Functional analysis of GRK5 variations in zebrafish.

(a) Whole mount in situ hybridisation of 20–22 somite stage zebrafish embryos for the leftward gene southpaw (spaw). Embryos were either left uninjected (NI) or were injected with a 5 basepair mismatch control MO (CMO) or a translation blocking MO against Grk5l, respectively. To analyse the functionality of the different Grk5l variants, fertilised eggs were simultaneously injected with capped RNAs for wild-type Grk5l or one of the four variants. Control embryos predominantly express spaw in the left lateral plate mesoderm (left panel), while. Grk5l depleted embryos show aberrant spaw expression bilaterally or right of the midline (middle and right panel). (b) Summary of 4 to 17 independent experiments with 114–432 embryos: embryos were scored for spaw expression left (L) or right (R) from the midline, for bilateral (B) or absent expression (A) See supplement for injections of RNA only. (c) WMISH of 48 hours post fertilisation (hpf) zebrafish embryos for the insulin gene to detect placement of the pancreas upon rescue attempts with Grk5l variant RNAs. In controls, pancreas placement is predominantly on the right side of the midline (indicated by dashed line), while Grk5l depletion renders embryos with left-sided pancreas. (d) Summary of 3 to 6 independent experiments with 42–133 embryos: pancreas position is indicated by L (left) or R (right). See supplement for injections of RNA only. (e) WMISH of 48 hpf zebrafish embryos for cardiac myosin light chain 2 (cmlc2) to monitor heart looping. Left panel shows a correctly looped heart (D), the middle displays an unlooped heart (N) and the right panel shows an inversely looped heart (L). (d) Summary of 3 to 6 independent experiments with 42–133 embryos scored for heart looping after co-injection of Grk5l MO and different rescue RNAs. See supplement for injections of RNA only. In all panels anterior to the top. Fisher’s exact test was applied to compare efficiency of rescue of variant to wt Grk5l and p values are indicated to the right.