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Multiplexed analysis of the secretin-like GPCR-RAMP interactome

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Science Advances  18 Sep 2019:
Vol. 5, no. 9, eaaw2778
DOI: 10.1126/sciadv.aaw2778
  • Fig. 1 Schematic of SBA assay procedure.

    (A) The GPCRs were epitope-fusion tagged at their N-terminal and C-terminal tails with HA and/or 1D4, respectively. The three RAMPs were tagged at their N-terminal and C-terminal tails with FLAG and OLLAS, respectively. (B) Unique Abs were coupled to different bar-coded beads to create an SBA with 70 different populations of capture beads, and the beads were subsequently pooled. DNA constructs encoding each of three epitope-tagged RAMPs and 23 epitope-tagged GPCRs were cotransfected in HEK293F cells such that all possible combinations of GRCPs and RAMPs were represented. The cells were solubilized in a detergent solution, which resulted in heterogeneous mixtures of solubilized proteins including the RAMPs, GPCRs, and putative GPCR-RAMP complexes. An aliquot of each cell lysate was incubated with an aliquot of SBA. Four identical assay plates were prepared in this manner. Following wash steps, a different PE-conjugated anti-epitope tag detection mAb was added to each of the four plates. (C) A Luminex FlexMap 3D instrument was used to measure the reporter fluorescence produced by the PE-conjugated detection mAb while simultaneously reading the bar code of each bead. From a single well, the specificity of RAMP Abs and GPCR Abs could be determined. Simultaneously, GPCR-RAMP complexes could be detected using either anti-epitope tag Abs, anti-GPCR Abs, or anti-RAMP Abs.

  • Fig. 2 Capture and detection of GPCR-RAMP complexes using anti-epitope tag mAbs.

    Lysates from cells transfected with each epitope-tagged RAMP construct and cotransfected with each epitope-tagged GPCR construct were incubated with the SBA, which included beads that were conjugated to mAbs targeting the four tags. Complexes were captured in multiplex fashion using one of the four mAbs. There are eight possible capture-detection schemes. The GPCR is captured using anti-1D4 mAb, and the GPCR-RAMP complex is detected using (A) PE-conjugated FLAG mAb or (B) PE-conjugated anti-OLLAS mAb. The GPCR is also captured using anti-HA mAb, and the GPCR-RAMP complex is detected using (C) PE-conjugated anti-FLAG mAb or (D) PE-conjugated anti-OLLAS mAb. The RAMP is captured using anti-FLAG mAb, and the GPCR-RAMP complex is detected using (E) PE-conjugated 1D4 mAb or (F) PE-conjugated anti-HA mAb. The RAMP is captured using anti-OLLAS mAb, and the GPCR-RAMP complex is detected using (G) PE-conjugated 1D4 mAb or (H) PE-conjugated anti-HA mAb. Data are presented for each of the three RAMPs. GPCR names are listed at the bottom of each RAMP panel, and the boxes are color coded. The occasional gray box indicates that the GPCR did not have the appropriate epitope tag to be captured or detected. The labels in bold correspond to secretin-like GPCRs. Data are median fluorescence intensity (MFI) and represent measurements obtained from at least three experiments performed in duplicate. The box plots represent the maximum and minimum extents of the measured values, and all data points are graphed.

  • Fig. 3 Validation of Abs used to capture GPCRs.

    To validate anti-GPCR Abs, lysates from cells transfected with each epitope-tagged GPCR construct (HA and/or 1D4) were incubated with the SBA, which included beads conjugated with 55 Abs targeting 21 GPCRs. (A) PE-conjugated anti-HA and (B) PE-conjugated anti-1D4 were used to detect any GPCRs captured by the beads. GPCRs are shown in alphabetical order, and the labels in bold correspond to secretin-like GPCRs. Each dot in each bee-swam plot represents a data point from one experiment. The blue dots indicate the signal from lysates containing the intended GPCR target, while gray dots indicate the signal from lysates containing another epitope-tagged GPCR target. Data are MFIs and representative of at least 200 experiments, each performed in duplicate. The occasional gray box indicates that the GPCR did not have the appropriate epitope tag to be captured or detected. At a statistical significance of P ≤ 0.05, we validated a total of 31 capture Abs, with at least one capture Ab for 19 of the 21 GPCRs studied. Validated Abs are underlined. Bead ID numbers are listed after each GPCR name, and the corresponding Ab name is provided in table S1.

  • Fig. 4 Graphical summary of GPCR-RAMP complexes detected using an SBA assay.

    Each circle depicts a unique GPCR along with each of the three RAMPs. Each GPCR is labeled and color coded. RAMP1 is colored gray, RAMP2 is colored lime, and RAMP3 is colored tangerine. Curved lines within the circles show GPCR-RAMP interactions, and the thicknesses of the lines show relative statistical significance (see below). The small labels around the circumference indicate the Abs used for the SBA experiments. In total, 4 anti-epitope tag Abs, 31 validated Abs to the 23 GPCRs included in the study, and 5 validated Abs to the three RAMPs are shown. Three GPCRs tested (CCR5, CCR7, and CXCR4) did not form complexes with RAMPs and are not shown here. The statistical significance derived for the particular interaction using the indicated capture-detection pair is represented by the thickness of the curved lines. P ≤ 0.05 is given an arbitrary thickness of 1, P ≤ 0.01 a thickness of 2, P ≤ 0.001 a thickness of 3, and P ≤ 0.0001 a thickness of 4. Underlined GPCR and RAMP names indicate previously unidentified GPCR-RAMP complexes. Bead ID numbers are listed with each GPCR name, and the corresponding validated Ab names are provided in table S1.

  • Fig. 5 Validation of RAMP-GPCR complex formation in cell membranes using PLA.

    Cells were cotransfected with epitope-tagged GPCR and RAMP2 and then incubated with α-HA and α-FLAG Abs. PLA was then carried out to quantitate GPCR-RAMP2 interactions. The number of PLA puncta per cell for each Z-stack captured was measured. Each Z-stack is of a different field of view. (A) Quantitation of control PLA experiments performed on CALCRL + RAMP2–cotransfected cells. PLA puncta counts were compared between samples that received both primary Abs and samples that received only anti-HA Ab, only anti-FLAG Ab, no primary Abs, or mock transfection with both primary Abs. Data are from two experiments performed with at least three replicates. (B) PLA puncta counts for cells cotransfected with RAMP2 and selected GPCRs. Data are from at least three experiments performed with at least five replicates. (C) Representative images of cells cotransfected with RAMP2 and selected GPCRs and subjected to PLA. Top row shows maximum projection of Z-stack, which is the maximum signal intensity for each channel at each point across all slices in the Z-stack. The bottom row shows snapshots from puncta quantification performed in Imaris. Scale bars, 5 μm (top row), 8 μm (bottom row). Blue, DAPI (4′,6-diamidino-2-phenylindole); red, PLA puncta; gray, Imaris spots. The statistical test for significance used was one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test (****P < 0.0001; NS, not significant).

  • Table 1 List of 23 GPCRs included in this study.

    Listed are the common abbreviations of each GPCR, GPCR family designation, and engineered epitope tag on the N-terminal and C-terminal tails. GPCRs are listed in alphabetical order. Secretin family receptors are shown in bold. Y, yes; N, no.

    GPCRAbbreviationGPCR familyN-terminal HA epitope tagC-terminal 1D4 epitope tag
    Atypical chemokine receptor
    3/C-X-C chemokine receptor 7
    ACKR3/CXCR7RhodopsinYY
    Pituitary adenylate
    cyclase-activating
    polypeptide type 1
    ADCYAP1R1SecretinYY
    Adhesion G protein–coupled
    receptor F5
    ADGRF5AdhesionYY
    Calcitonin receptor-like
    receptor
    CALCRLSecretinYY
    Calcitonin receptorCALCRSecretinYY
    C-C chemokine receptor
    type 5
    CCR5RhodopsinNY
    C-C chemokine receptor
    type 7
    CCR7RhodopsinNY
    Corticotropin-releasing
    hormone receptor 1
    CRHR1SecretinYY
    Cortocotropin-releasing
    hormone receptor 2
    CRHR2SecretinYY
    C-X-C chemokine receptor
    type 3
    CXCR3RhodopsinYN
    C-X-C chemokine receptor
    type 4
    CXCR4RhodopsinNY
    Gastric inhibitory
    polypeptide receptor
    GIPRSecretinYY
    Glucagon receptorGCGRSecretinYY
    Growth hormone releasing
    hormone
    GHRHRSecretinYY
    Glucagon-like receptor 1GLP1RSecretinYY
    Glucagon-like receptor 2GLP2RSecretinYY
    G protein–coupled receptor 4GPR4RhodopsinYN
    G protein–coupled receptor 182GPR182RhodopsinYN
    Parathyroid hormone
    receptor 1
    PTH1RSecretinYY
    Parathryoid hormone
    receptor 2
    PTH2RSecretinYY
    Secretin receptorSCTRSecretinYY
    VIP and PACAP receptor 1VIPR1SecretinYY
    VIP and PACAP receptor 2VIPR2SecretinYY
  • Table 2 Length of native signal sequence of each secretin-like GPCR and ADGRF5.

    GPCRSignal sequence
    ADCYAP1R11–23
    ADGRF51–21
    CALCRL1–22
    CALCR1–42
    CRHR11–23
    CRHR21–19
    GIPR1–25
    GCGR1–26
    GLP1R1–23
    GLP2RNone
    GHRHR1–22
    PTH1R1–28
    PTH2R1–24
    SCTR1–27
    VIPR11–30
    VIPR21–20

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaaw2778/DC1

    Supplementary Methods

    Fig. S1. Coexpression of GPCR clusters with RAMPs and the position of selected GPCRs on the phylogenetic tree.

    Fig. S2. Validation of anti-epitope tag mAbs to capture and detect engineered RAMPs and GPCRs.

    Fig. S3. Validation of Abs used to capture RAMPs.

    Fig. S4. Analysis of anti-GPCR Ab cross-reactivity.

    Fig. S5. Detection of GPCR-RAMP complexes following capture by all anti-GPCR Abs.

    Fig. S6. Statistical validation of GPCR-RAMP SBA datasets.

    Fig. S7. Detection of CALCRL-RAMP2 interactions in cell membranes using PLA.

    Table S1. The ID of the bead coupled to each specific Ab, the source of the Ab, and the product code.

    Table S2. Statistical significances of complex formation between each GPCR and RAMP complex pair reported as P values.

    Table S3. Overall statistic for GPCR-RAMP complex formation.

    Table S4. Statistical metrics used to compare GPCR-RAMP complex formation datasets.

    References (4143)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Methods
    • Fig. S1. Coexpression of GPCR clusters with RAMPs and the position of selected GPCRs on the phylogenetic tree.
    • Fig. S2. Validation of anti-epitope tag mAbs to capture and detect engineered RAMPs and GPCRs.
    • Fig. S3. Validation of Abs used to capture RAMPs.
    • Fig. S4. Analysis of anti-GPCR Ab cross-reactivity.
    • Fig. S5. Detection of GPCR-RAMP complexes following capture by all anti-GPCR Abs.
    • Fig. S6. Statistical validation of GPCR-RAMP SBA datasets.
    • Fig. S7. Detection of CALCRL-RAMP2 interactions in cell membranes using PLA.
    • Table S1. The ID of the bead coupled to each specific Ab, the source of the Ab, and the product code.
    • Table S2. Statistical significances of complex formation between each GPCR and RAMP complex pair reported as P values.
    • Table S3. Overall statistic for GPCR-RAMP complex formation.
    • Table S4. Statistical metrics used to compare GPCR-RAMP complex formation datasets.
    • References (4143)

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