β2 Adrenergic Receptor Fluorescent Protein Fusions Traffic to the Plasma Membrane and Retain Functionality

Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β2 adrenergic receptor (β2AR). We have characterized these β2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β2ARR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality

In Vitro Mutational Analysis of the β2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor

Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs

β₂ Adrenergic Receptor Fluorescent Protein Fusions Traffic to the Plasma Membrane and Retain Functionality

<div><p>Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β₂ adrenergic receptor (β₂AR). We have characterized these β₂AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β₂AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β₂AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality.</p> </div

<i>bam</i> transgenic constructs.

<p>(A) Diagrams of ovariole tip and (B) testis tip of wildtype flies. GSCs differentiate into cystoblasts (CB, ovariole) or gonialblasts (GB, testis), which undergo four synchronous, mitotic divisions. In females, Bam expression (yellow) is restricted to the CB, 2-,4-, and 8-cell cysts. In males, Bam expression occurs in 4-,8-, and 16-cell cysts. Somatic cells/somatic stem cells are shown in pink, germ cells in blue and yellow (when expressing Bam), GSCs in light blue, and spectrosomes (in GSCs) and fusomes (in cysts) in red. (C) <i>bam</i> transgenic constructs. All constructs are drawn to scale and contain the entire <i>bam</i> open reading frame (thick bars), 2 small introns, and non-coding regions (thin bars). Green color corresponds to <i>D</i>. <i>melanogaster</i> sequences, orange to <i>D</i>. <i>simulans</i> sequences, and yellow to the YFP coding sequence. ATG denotes the start codon, and 5’ and 3’ UTR sequence boundaries are from <i>D</i>. <i>melanogaster</i> genome release v. 5.30 (Flybase) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005453#pgen.1005453.ref106" target="_blank">106</a>]. The transcription start site is denoted as +1 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005453#pgen.1005453.ref021" target="_blank">21</a>] and the poly(A) addition sequence is denoted as A [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005453#pgen.1005453.ref023" target="_blank">23</a>].</p

<i>sim-bam-yfp; bam</i>⁻ovaries have multiple defects.

<p>(A-B) <i>mel-bam-yfp; bam</i>⁻ovaries show wildtype morphology including proper Bam-YFP expression, correct number of GSCs identified by spectrosomes, and proper numbers of cells/cyst. (C-D) <i>sim-bam-yfp; bam</i>⁻ovaries show reduced number of GSCs (*) and contain egg chambers with improper number of cells/cyst. (E) <i>D</i>. <i>melanogaster bam</i> null mutant shows “bag of marbles” phenotype. (A-E) Ovaries are from flies aged 3–5 days post-eclosion and stained with antibodies to Vasa (green), Hts-1B1 (red), and YFP (blue). Scale bar, 50μm. (F) Average GSC number across different genotypes. N = 50 ovarioles. (<i>t</i>-test, ***<i>P</i><0.001).</p

<i>Wolbachia</i> increases the fertility of <i>D</i>. <i>melanogaster bam</i> hypomorphs without altering <i>bam</i> RNA levels.

<p>(A) One female and two tester males were allowed to mate and the trio was removed from the vial after 8 days. Fertility is shown as the average number of progeny per female +/- SEM for each vial. N = 20. <i>Wolbachia</i>-infected (<i>w</i>Mel) <i>bam</i> hypomorphs are significantly more fertile than uninfected <i>bam</i> hypomorphs, <i>bam</i>-Tet (<i>t</i>-test, ***<i>P</i><0.001). (B) qRT-PCR of ovarian mRNA from <i>D</i>. <i>melanogaster bam</i> hypomorphs with and without <i>Wolbachia</i>. The <i>D</i>. <i>melanogaster</i> marker strain <i>y w</i> (grey, two wildtype copies of <i>bam</i>) is shown for reference. There is no statistical difference in <i>bam</i> expression of the <i>bam</i> hypomorph with and without <i>Wolbachia</i> (<i>P</i> = 0.253; <i>t</i>-test).</p

<i>sim-bam-yfp</i> rescues <i>D</i>. <i>melanogaster bam</i> mutant male sterility.

<p>(A) <i>mel-bam-yfp</i> and <i>sim-bam-yfp</i> both rescue male sterility under standard fertility conditions. One male and two tester females were allowed to mate and the trio was transferred to a new vial every five days. No comparisons are significantly different. N ranged between 42 and 46 males at start of experiment; due to mortality N ranged between 37 and 43 at end of experiment. (B) <i>sim-bam-yfp</i> but not <i>mel-bam-yfp</i> rescues male sterility under sperm exhaustion conditions. One male was allowed to mate with a new pair of virgin tester females everyday for five days. Male fertility is the average number of progeny per male +/- SEM for each vial. N ranged between 28 and 33 males at start of experiment; due to mortality N ranged between 22 and 28 at end of experiment. Transgenes are inserted in attP40. (<i>t</i>-test, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p

Analysis of <i>bam</i> RNA and protein expression in transgenic lines and control strains.

<p>(A) Underexpression of <i>bam</i> RNA in <i>mel-bam-yfp; bam</i>⁻ovaries is not due to genetic background or the YFP tag. Ovarian <i>bam</i> RNA levels from <i>mel-bam-yfp</i> (red) and <i>sim-bam-yfp</i> (blue) in the <i>bam</i> mutant background (<i>bam</i>^<i>Δ86</i><i>/bam</i>^<i>Δ59</i>), and the <i>D</i>. <i>melanogaster bam</i> heterozygote (<i>+</i>/<i>bam</i>^<i>Δ59</i>, green). <i>bam</i> levels of <i>mel-bam-yfp</i> in a different <i>bam</i> mutant background (<i>bam</i>^<i>Δ86</i><i>/bam</i>^<i>BG</i>) (orange) and of a different <i>bam</i> transgene (yellow, <i>bam</i>-α; <i>bam</i>^<i>–</i>) are also reduced relative to the <i>bam</i> heterozygote. ΦC31-integrated transgenes in (A) are docked in attP40. (B) Transgene expression is stable across different insertion sites. We compared <i>bam</i> RNA levels from <i>mel-bam-yfp; bam</i>⁻and <i>sim-bam-yfp; bam</i>⁻ovaries in two different insertion sites, attP40 and attP16a. The <i>bam</i>⁻genotype is <i>bam</i>^Δ86/<i>bam</i>^Δ59 as explained in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005453#pgen.1005453.t001" target="_blank">Table 1</a>. (C) <i>bam</i> expression levels show little variation across strains. <i>bam</i> RNA levels were compared between the <i>D</i>. <i>melanogaster bam</i> heterozygote shown in (A) to that of various wildtype or marker lines (Canton S [CS], <i>y w</i>, and <i>y; cn bw; sp</i>) that were made heterozygous over a <i>D</i>. <i>melanogaster bam</i> mutant (<i>bam</i>^<i>Δ59</i>). The <i>bam</i> sequence in <i>mel-bam-yfp</i> was cloned from <i>y; cn bw; sp</i>. For A-C, N = 3 biological replicates for each sample. Significance was determined by <i>t</i>-test, * <i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. No significant expression differences were found in (C). (D) Western blot comparing sim-Bam-YFP and mel-Bam-YFP levels. 20μg of total protein was loaded into each lane. Western blot probed with anti-YFP or anti-α-Tubulin antibodies.</p

sim-Bam maintains interactions with mel-Bgcn in immunoprecipitates from S2 cells.

<p>(A-B) Control experiments with mel-Bam and mel-Bgcn. (A) Cells were transfected with mel-Bam::HA and either mel-Bgcn::MYC or MYC. Anti-MYC immunoprecipitates were analyzed by Western blot. (B) Cells were transfected with mel-Bgcn::MYC and either mel-Bam::HA or HA. Anti-HA immunoprecipitates were analyzed by Western blot. (C-D) IP experiment with sim-Bam and mel-Bgcn. (C) Cells were transfected with sim-Bam::HA and either mel-Bgcn::MYC or MYC. Anti-MYC immunoprecipitates were analyzed by Western blot. (D) Cells were transfected with mel-Bgcn::MYC and either sim-Bam::HA or HA. Anti-HA immunoprecipitates were analyzed by Western blot. Gels are loaded with 25% of total input (Input), 100% of immunoprecipitate (IP), and 10% of protein that did not immunoprecipitate (flow through, FT). (E-F) Ovaries of <i>sim-bam-yfp;bam</i>^<i>−</i>flies show a varying range of ovarian defects with mild (E) and moderate (F) examples shown for comparison. (G-H) Removal of a copy of <i>bgcn</i> (<i>bgcn</i>^<i>1</i>) does not enhance the range of phenotypes seen in <i>sim-bam-yfp;bam</i>^<i>−</i>ovaries. No tumorous ovaries were seen (N > 50 ovarioles). (E-H) Ovaries are stained with antibodies to Vasa (green) and Hts-1B1 (red). Scale bar, 50μm.</p