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 and Bioinformatics Analysis of the M71 Odorant Receptor and Its Superfamily

We performed an extensive mutational analysis of the canonical mouse odorant receptor (OR) M71 to determine the properties of ORs that inhibit plasma membrane trafficking in heterologous expression systems. We employed the use of the M71::GFP fusion protein to directly assess plasma membrane localization and functionality of M71 in heterologous cells in vitro or in olfactory sensory neurons (OSNs) in vivo. OSN expression of M71::GFP show only small differences in activity compared to untagged M71. However, M71::GFP could not traffic to the plasma membrane even in the presence of proposed accessory proteins RTP1S or mβ2AR. To ask if ORs contain an internal “kill sequence”, we mutated ~15 of the most highly conserved OR specific amino acids not found amongst the trafficking non-OR GPCR superfamily; none of these mutants rescued trafficking. Addition of various amino terminal signal sequences or different glycosylation motifs all failed to produce trafficking. The addition of the amino and carboxy terminal domains of mβ2AR or the mutation Y289A in the highly conserved GPCR motif NPxxY does not rescue plasma membrane trafficking. The failure of targeted mutagenesis on rescuing plasma membrane localization in heterologous cells suggests that OR trafficking deficits may not be attributable to conserved collinear motifs, but rather the overall amino acid composition of the OR family. Thus, we performed an in silico analysis comparing the OR and other amine receptor superfamilies. We find that ORs contain fewer charged residues and more hydrophobic residues distributed throughout the protein and a conserved overall amino acid composition. From our analysis, we surmise that it may be difficult to traffic ORs at high levels to the cell surface in vitro, without making significant amino acid modifications. Finally, we observed specific increases in methionine and histidine residues as well as a marked decrease in tryptophan residues, suggesting that these changes provide ORs with special characteristics needed for them to function in olfactory neurons

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

FUNCTIONAL EVOLUTION OF THE BAG OF MARBLES GENE IN DROSOPHILA

190 pagesUnderstanding how organisms adapt to changes in their environment at both the genotypic and phenotypic level is a fundamental challenge of evolutionary biology. In Drosophila melanogaster a key germline stem cell differentiation factor which is required for gametogenesis, bag of marbles (bam) shows signatures of adaptive evolution between its sibling species D. simulans, but not in several outgroup species. However, the functional consequences and drivers of the genetic signals of adaptation at bam remain unclear. Bam is a novel protein to a dipteran lineage containing Drosophila, therefore it is possible bam has functionally diversified across the Drosophila genus, only gaining its known function in the lineage leading to D. melanogaster and D. simulans, thereby resulting in adaptive evolution for this function. Additionally, it is possible that bam quickly evolved a role in GSC differentiation in Drosophila, but that function has been shaped by germline conflicts in specific lineages. Notably, the endosymbiotic bacteria Wolbachia resides in the germline and rescues the fertility defect of a bam partial loss-of-function mutant in D. melanogaster. In this dissertation I asked if bam function is conserved across five representative Drosophila species to test if a novel role in germline stem cell differentiation could be driving the adaptive evolution of bam. I also asked if Wolbachia variation impacts the rescue of the bam partial loss-of- function phenotype to further understand the dynamics of the bam and Wolbachia interaction in D. melanogaster. I found that bam function is not fully conserved across these species, and its function may evolve on a relatively short time scale. I also found an effect of Wolbachia variation on the degree of the bam fertility rescue. My findings indicate that the episodic signals of adaptive evolution at bam are unlikely to be driven by a single gain in function for bam as a GSC differentiation factor, and that we can use variation in Wolbachia to further define the biological mechanism of its interaction with bam to understand how Wolbachia impacts bam evolution

<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

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>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