A Comprehensive Mutagenesis Screen of the Adhesion GPCR Latrophilin-1/ADGRL1

Abstract

Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse cellular processes in neurobiology, development, immunity, and numerous diseases. The lack of molecular understanding of their activation mechanisms, especially with regard to the transmembrane domains, hampers further studies to facilitate aGPCR-targeted drug development. Latrophilin-1/ADGRL1 is a model aGPCR that regulates synapse formation and embryogenesis, and its mutations are associated with cancer and attention-deficit/hyperactivity disorder. Here, we established functional assays to monitor latrophilin-1 function and showed the activation of latrophilin-1 by its endogenous agonist peptide. Via a comprehensive mutagenesis screen, we identified transmembrane domain residues essential for latrophilin-1 basal activity and for agonist peptide response. Strikingly, a cancer-associated mutation exhibited increased basal activity and failed to rescue the embryonic developmental phenotype in transgenic worms. These results provide a mechanistic foundation for future aGPCR-targeted drug design.

Keywords: Membrane Architecture; Molecular Biology; Protein Structure Aspects.

Graphical abstract

Figure 1

Lphn1 and Lphn3 Decrease cAMP Level and Increase SRE Level in Transfected Cells (A) Schematic domain diagram of Lphns as a model aGPCR. All aGPCRs have a GAIN domain, a TM domain, and variable other domains. Stachel peptide is a tethered agonist. Yellow line indicates Stachel peptide and * indicates cleavage site. (B) β2-adrenergic receptor assay to detect cAMP signaling of rat Lphn1 and human Lphn3 in transfected HEK293 cells. Lphn inhibits ISO-induced cAMP elevation. Signaling data are obtained from n = 3 independent experiments performed in triplicates and represented as means ± SEM. cAMP level was measured by GloSensor assay. Figure modified from Li et al. (2018). SEM, standard error of the mean. (C) Forskolin assay to detect cAMP signaling of Lphn1 and Lphn3 in transfected HEK293 cells. Lphn1 inhibits forskolin-induced cAMP elevation. (D) Basal activity of Lphn1 and Lphn3 as measured by the SRE-luciferase reporter assay. ^NSp > 0.05; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001.

Figure 2

Stachel Peptide is a Tethered Agonist for Lphn1 (A) Schematic diagram for Lphn1 constructs encoding full-length Lphn1, inactive-like TM domain of Lphn1 (TM), or active-like Stachel peptide/TM domain of Lphn1 (P + TM). The constructs are FLAG-tagged at the indicated positions. Stachel peptide is colored yellow. Asterisk represents autoproteolysis site within the GAIN domain. (B) Sequence alignment of Stachel peptides from different Lphns. Identical residues are highlighted in gray. (C) Signaling activity of Lphn1 constructs from (A) as measured by the β2-adrenergic receptor cAMP assay. Cells were pre-incubated with or without pertussis toxin and treated with 100 μM synthetic agonist peptide or solvent. Data are plotted as percentage of isoproterenol response of empty vector-transfected cells. Data are represented as mean ± SEM of three independent experiments, three repeats each (n = 9). SEM, standard error of the mean. (D) Signaling activity of Lphn1 constructs as in (C) in the forskolin cAMP assay. Data are plotted as percentage of forskolin response of empty vector-transfected cells. Data are obtained from one typical experiment performed in triplicate and represented as mean ± SEM (n = 3). ^NSp > 0.05; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. NS, Not Significant.

Figure 3

TM Mutations That Affect Basal Activity of Lphn1 (A–I) TM mutations that have an effect on the basal activity of full-length Lphn1 are shown for the cAMP signaling assay (A, D, and G), SRE signaling assay (B, E, and H), and cell-surface expression quantification (C, F, and I). The mutations are categorized as: (A–C) mutations that map to the indicated conserved motifs from rhodopsin, secretin, and adhesion families; (D–F) mutations that lead to constitutive activity; and (G–I) previously reported cancer-associated mutations that affect Lphn1 signaling. Mutations that abolish receptor response to the agonist peptide are shown in Figure 5. One mutation may belong to more than one category. See Table S1 for raw data and for other mutants that had no effect. See Figure 4 for structural visualization of basal activity mutants. See Figures S4 and S5 for raw cell-surface expression data. Mutations that are introduced into transgenic worm are indicated by a cyan star. Basal activity in cAMP assay was detected as percent of wild-type (WT) Lphn1 after activation with 50 nM isoproterenol. The effect of the agonist peptide was detected by pre-incubation with 100 μM synthetic agonist peptide for 5 min before isoproterenol activation. Signaling data are obtained from three independent β2AR co-expression experiments performed in triplicates and represented as means ± SE Basal activity in SRE assay was normalized to empty vector-transfected cells. Signaling data are obtained from three independent SRE experiments performed in triplicates and presented as means ± SE Cell-surface expression for each mutant was obtained from three independent flow cytometry experiments using the same cells as those used for the cAMP assay and presented as mean ± SE Additional DNA titration experiments for some of the mutants with very low or high expression levels are presented on the right side of the panels and were aimed to measure receptor signaling at expression levels comparable to WT. SE, standard error. ^NSp > 0.05; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. NS, Not Significant.

Figure 4

Basal Activity Mutations Mapped on the Modeled TM Structure of Lphn1 (A) Snake plot for visualization of basal activity mutations on the transmembrane helices. (B) All basal activity mutations mapped on the Lphn1 TM domain, which is modeled based on the inactive CRFR structure (PDB ID: 4K5Y). Mutations that increase basal activity are colored red, mutations that decrease basal activity are colored green, and mutations that increase basal activity and also decrease response to the agonist peptide are colored blue. (C) A cytoplasmic view of the modeled Lphn1 structure showing the basal activity mutants and the mutants that map to conserved region homologous to the NPxxY motif. (D) A view of the modeled Lphn1 structure showing the basal activity mutants that map to conserved regions homologous to the DRY motif and the ionic lock. I1045N mutation (colored magenta) affects peptide response. (E) Cancer-associated mutations on Lphn1 TM domain that affect signaling. V1094I (V1095I in human Lphn1 in upper aerodigestive tract carcinoma), R885L (R886L in human Lphn1 in endometrioid carcinoma), C1098Y (C1098Y in human Lphn1 in endometrioid carcinoma), Y1001F (Y1019F in human Lphn3 in lung adenocarcinoma), and I1045N (I1146N in human Lphn1 in hepatocellular carcinoma) were reported previously (Kan et al., 2010, O'Hayre et al., 2013) (colored yellow). Mutations that are introduced into transgenic worm are labeled with a cyan star.

Figure 5

TM Mutations That Affect Response of Lphn1 to Agonist Peptide (A–C) TM mutations that have an effect on response of full-length Lphn1 to the agonist peptide are shown in the cAMP signaling assay (A), SRE signaling assay (B), and cell-surface expression quantification (C). One mutation may belong to more than one category and might be listed in Figure 3 as well. Mutations that are introduced into transgenic worm are indicated by a cyan star. See Table S1 for raw data and for other mutations that had no effect. See Figure 6 for structural visualization of peptide response mutations. See Figures S4 and S5 for cell-surface expression data. See Figure 3 legend for details about the cAMP assay, SRE assay, and cell-surface expression quantification. ^NSp > 0.05; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. NS, Not Significant.

Figure 6

Peptide Response Mutations Mapped on the Modeled TM Structure of Lphn1 (A) Snake plot for visualization of peptide response mutations on the transmembrane helices. (B) All mutations that affect response of the receptor to the agonist peptide mapped on the Lphn1 TM domain, which is modeled based on the peptide-bound active GLP receptor structure (PDB ID: 5VAI). Mutations that affect peptide response are colored magenta, mutations that affect both peptide response and basal activity are colored blue, and mutations that likely affect peptide response are colored orange. (C) Superimposition of the model of Lphn1 TM domain (gray) with the GLP1 peptide (red)-bound GLP1 receptor/G protein (green/tan) complex structure (PDB ID: 5VAI). Lphn1 residues are colored as in B. (D) Close-up extracellular view of (C) showing the modeled Lphn1 TM domain and the GLP1 peptide. Peptide response mutants that map to the extracellular side of the receptor as well as GLP peptide residues important for its interaction with the GLP receptor are shown by sticks. (E) Close-up cytoplasmic view of (C) showing the modeled Lphn1 TM domain and the G protein. Peptide response mutants that map to the cytoplasmic side of the receptor are shown by sticks. Mutations that are introduced into transgenic worms are labeled with cyan asterisk.

Figure 7

An Overactive Cancer-Associated Mutant Fails to Rescue the Developmental Phenotype of LAT-1 Knockdown in C. elegans (A) Expression and protein localization of all three point mutation-containing variants is indistinguishable from wild-type lat-1::gfp. Fluorescence images show presence of LAT-1 and the variants at the plasma membrane of pharyngeal muscle cells, neurons in the nerve ring, and the pharyngeal nervous system. Scale bars represent 10 μm. (B and C) The point mutations within the TM of LAT-1 lead to different abilities to rescue fertility (brood size, B) and lethality (individuals reaching adulthood, C) of lat-1 mutants. All three variants ameliorate the fertility defects observed in lat-1 mutants similar to a wild-type lat-1 transgene or a construct comprising the extracellular region tethered to the membrane via the first TM (lat-1 [aa1–581]) (B). Only LAT-1(L790A) does not rescue lethality, while LAT-1(F763A) and LAT-1(H792A) display the same functionality as a wild-type lat-1 transgene. Data are shown as percentage of the original brood sizes (C). The wild-type lat-1 transgene, which rescues fertility and lethality, and a lat-1(aa1–581) construct, which only rescues fertility, served as controls. Data are shown as means ± SEM, n ≥ 20, n.s., not significant; **p < 0.01; ***p < 0.001. SEM, standard error of the mean.