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G protein-coupled receptors (GPCRs) are found in all eukaryotic cells examined to date where they function as membrane-bound proteins that bind a multitude of extracellular ligands to initiate intracellular signal transduction systems controlling cellular physiology. GPCRs have seven heptahelical membrane spanning domains connected by extracellular and intracellular loops with an extracellular N-terminus and an intracellular C-terminus. The N-terminus has been the least studied domain of most GPCRs. The yeast Ste2p protein, the receptor for the thirteen amino acid peptide pheromone α-factor, has been used extensively as a model to study GPCR structure and function. In this study we constructed a number of deletions of the Ste2p N-terminus and uncovered an unexpected function as a negative regulatory domain. We examined the role of the N-terminus in expression, signaling function and ligand-binding properties and found that the residues 11-30 play a critical role in receptor expression on the cell surface. The studies also indicated that residues 2-10 of the N-terminus are involved in negative regulation of signaling as shown by the observation that deletion of these residues enhanced mating and gene induction. Furthermore, our results indicated that the residues 21-30 are essential for optimal signaling. Overall, we propose that the N-terminus of Ste2p plays multiple regulatory roles in controlling receptor function.
G protein-coupled receptors (GPCRs) represent the largest family of cell surface molecules involved in signal transduction in humans. Possessing about 800 GPCR-encoding genes in the human genome, these proteins control numerous physiological processes, such as vision, taste, smell, neurotransmission, cardiovascular, endocrine, immune responses, and reproductive functions. Dysfunction of these receptors is associated with many diseases or pathological conditions such as obesity, blindness, cancer, etc. [1–5]. Hence elucidating the mechanism of signal transduction by these receptors is indispensable to understand and treat many diseases.
All GPCRs share a common structural organization with an extracellular N-terminus, seven transmembrane domains connected by extracellular and intracellular loops, and a cytoplasmic C-terminus [6–8]. Despite the diversity of their ligands and a lack of strong sequence similarity, the underlying mechanisms of signal transduction are similar, as GPCRs couple the binding of ligands to the activation of specific heterotrimeric guanine nucleotide-binding proteins (G proteins) and/or non-G protein signaling molecules leading to the modulation of downstream effector proteins and gene expression [9–11]. The mechanism of signal transduction by GPCRs to their cognate heterotrimeric G-proteins is still not fully elucidated at the atomic level.
Although GPCRs contain several distinct domains, the majority of studies have focused on the transmembrane helices. However, a number of studies indicate that the N-terminus also plays an important role in receptor function [6, 12, 13]. An N-terminal network of three disulfide bonds in Class B secretin receptors stabilizes the N-terminal structure, the alteration of which impairs ligand interactions . Likewise, a diverse variety of N-terminal domain motifs in the N-terminal domain of Class B adhesion receptors determine ligand specificity. The conserved N-terminal Venus flytrap domain in Class C glutamate receptors and N-terminal Wnt-binding domains in Frizzled/Smoothened receptors have been reported to regulate ligand binding and receptor activation [6, 8, 12, 13]. The N-terminal domains of protease-activated receptors (PARs) and glycoprotein hormone receptors (GpHRs) play an important role in their activation [15, 16]. Recently, the N-terminus of GPR56, an adhesion G protein-coupled receptor that plays a key role in cortical development, has been reported to constrain receptor activity . Truncation of the N-terminus of several GPCRs including CB1 cannabinoid , α1D adrenergic  and GPR37  has been shown to enhance cell surface expression.
The mating pheromone α-factor receptor Ste2p is a GPCR that initiates the mating pathway of the yeast Saccharomyces cerevisiae. This receptor-ligand system has been used as an attractive and appropriate model for understanding the mechanism of signal transduction by peptide-responsive GPCRs [21–24]. Though there has been considerable study of the extracellular and intracellular loops as well as the C-terminus and transmembrane domains of Ste2p, less is known about the role of the N-terminal domain in signaling. The N-terminus of Ste2p is ~48 amino acids long, and it harbors two glycosylation sites (N25 and N32) which were eliminated by mutation (N25A and N32A) without affecting receptor function . Other studies indicated that the N-terminus contributed to receptor dimerization [26, 27], and three residues (Pro 15, Ile24, and Ile29) were found to be essential for mating but not for signaling as measured by growth arrest and reporter gene (FUS1) activation assays [28, 29]. Truncation of parts of the N-terminus implicated this domain in cellular fusion (mating) during late stages of conjugation of opposite mating types .
In this study we performed deletion mutagenesis on the N-terminus and analyzed the mutant receptors by protein expression, ligand binding and signaling assays. The results showed that deletion of portions of the N-terminus affected the surface expression levels of the receptor. We also provide evidence that deletion of residues 2-10 results in enhanced signaling activities of the receptor, suggesting that the N-terminus of the full-length receptor is involved in negative regulation of signaling. Furthermore, removal of residues 21-30 affected signaling response drastically without changing the ligand binding affinity. Thus our results indicated that the N-terminus plays multiple regulatory roles in receptor function.
Saccharomyces cerevisiae strain LM102 [MATa ste2 FUS1-lacZ::URA3 bar1 ura3 leu2 his4 trp1 met1]  was used for growth arrest, FUS1-lacZ gene induction, mating, and saturation binding assays. Protease-deficient strain BJS21 [MATa, prc1-407 prb1-1122 pep4-3 leu2 trp1 ura3-52 ste2::KanR  was used for protein isolation to decrease receptor degradation during immunoblot analyses . The tryptophan-selectable plasmid pBEC2 with STE2 (Cys59Ser/Cys252Ser, termed the “Cys-less” receptor) and containing C-terminal FLAG™ and His-tags  was transformed by the method of Geitz . We refer to this receptor as the “wild-type” herein as it has been established in previous studies in our lab and others that this receptor is equivalent in activity to the naturally-occurring, wild type receptor [27, 32, 41–43]. Yeast transformants were selected by growth on yeast minimal medium  lacking tryptophan and supplemented with casamino acids (10g/L, Research Products International Corp., Prospect, IL.) designated as MLT to maintain selection for the plasmid. The cells were cultured in MLT and grown to mid log phase at 30°C with shaking (200 rpm) for all assays. S. cerevisiae strain TBR1 [MATα FLO11 ura3 his2 leu2]  was used as the opposite mating type (LM102 MATa) for mating assays.
Tritiated [3H] α-factor (9.33 Ci/mmol) prepared as previously described previously  was used in saturation binding assays on whole cells. Cells (LM102) expressing wild type or mutant Ste2p were harvested, washed three times with YM1 , and adjusted to a final concentration of 3 × 107cells/mL. Cells (600 μL) were combined with 150 μL of ice-cold 5× binding medium (YM1 plus protease inhibitors [YM1i]  supplemented with [3H]α-factor and incubated at room temperature for 30 min. The final concentration of [3H]α-factor ranged from 0.5 × 10−10 to 1 × 10−6 M. Upon completion of the incubation interval, 200 μL aliquots of the cell-pheromone mixture were collected in triplicate on glass fiber filter mats and washed for 5 seconds at room temperature with phosphate buffered saline, pH 7.4 using the Standard Cell Harvester (Skatron Instruments, Sterling, VA). Retained radioactivity on the filter was counted by liquid scintillation spectroscopy. Cells lacking Ste2p were used as a nonspecific binding control for the assays. Binding assays were repeated a minimum of three times, and similar results were observed for each replicate. Specific binding for each mutant receptor was calculated by subtracting the nonspecific values (radioactivity obtained from cells lacking receptor) from those obtained for total binding. Specific binding data were analyzed by nonlinear regression analysis for single-site binding using GraphPad Prism 5.04 (GraphPad Software, San Diego, CA) to determine the Kd and Bmax values for each mutant receptor.
BJS21 cells, expressing wild type or the N-terminal deletion mutants of Ste2p grown in MLT, were used to prepare total cell membranes isolated as previously described . Protein concentration was determined by the BioRad protein assay (BioRad, Hercules, CA), and membranes were solubilized in sodium dodecyl sulfate (SDS) sample buffer (10% glycerol, 5% 2-mercaptoethanol, 1% SDS, 0.03% bromophenol blue, 62.5 mM Tris, pH 6.8). Proteins were fractioned by SDS–PAGE (10% acrylamide with 5% stacking gel) along with pre-stained Precision Plus protein standards (BioRad) and transferred to an Immobilon™P membrane (Millipore Corp., Bedford, MA). The blot was probed with anti-FLAG™ M2 antibody (Sigma/Aldrich Chemical, St. Louis, MO), and bands were visualized with the West Pico chemiluminescent detection system (Pierce). The total intensity of all Ste2p bands in each lane was determined using a ChemiDoc XRS photodocumentation system with Quantity One one-dimensional analysis software (version 4.6.9, BioRad, Hercules, CA). Immunoblot experiments were repeated at least three times and yielded similar results. Constitutively-expressed membrane protein Pma1p was used as a loading control as described previously  using Pma1p antibody (Thermo Scientific, Rockford, IL).
Total membrane proteins prepared as described above were resuspended in sodium phosphate (50 mM, pH 7.5), supplemented with 500 units of glycerol-free PNGase F (New England Biolabs, Inc., Beverly, MA), and incubated at 37°C for 2 h. A negative control was run in parallel in which no enzyme was added prior to incubation at 37°C. Upon termination of the incubation interval, the membranes were pelleted by centrifugation (15,000 × g, 10 min), and the resulting pellet was dissolved in SDS sample buffer. The samples were used for FLAG immunoblot analysis as described above.
S. cerevisiae LM102 cells, expressing Cys-less “Wild type” Ste2p and deletion mutants, were grown at 30°C overnight in MLT, harvested, washed three times with water, and resuspended at a final concentration of 5 × 106cells/mL . Cells (1 mL) were combined with 3.5 mL of agar noble (1.1%) and poured as a top agar lawn onto a MLT medium agar plate. Filter disks (BD, Franklin Lakes, NJ) impregnated with α-factor (0.5, 0.25, 0.125, 0.06, 0.03 μg/disk) were placed on the top agar. The plates were incubated at 30°C for 24h and then observed for clear halos around the disks. The experiment was repeated at least three times, and reported values represent the mean of these tests.
LM102 cells, expressing Cys-less “Wild type” Ste2p and deletion mutants, were grown at 30°C in selective media, harvested, washed three times with fresh media and resuspended at a final concentration of 5 × 107 cells/mL. Cells (500 μl) were combined with α-factor (final concentration of 1.0 μM; this concentration is expected to saturate all the receptors on the surface) and incubated at 30°C for 90 min. The cells were transferred to a 96-well flat bottom plate (Corning Incorporated, Corning, NY) in triplicate, permeabilized with 0.5% Triton X-100 in 25 mM PIPES buffer (pH 7.2) and then β-galactosidase assays were carried out using fluorescein di-β-galactopyranoside (Molecular Probes, Inc., Eugene, OR) as a substrate as described previously [9, 40]. The reaction mixtures were incubated at 37°C for 60 min and 1.0 M Na2CO3 was added to stop the reaction. The fluorescence of the samples (excitation of 485 nm and emission of 530 nm) was determined using a 96-well plate reader Synergy2 (BioTek Instruments, Inc., Winooski, VT). The data were analyzed using Prism software (GraphPad Prism version 5.03 for Windows, GraphPad Software, San Diego CA). The experiments were repeated at least three times and reported values represent the mean of these tests. One-way ANOVA followed by Dunnett’s post hoc test was used to determine statistical significance (n = 4; p < 0.05).
S. cerevisiae MATa cells expressing various Ste2p constructs and MATα cells expressing wild type receptor were grown overnight at 30°C, harvested by centrifugation, resuspended in fresh YEPD, and counted using a hemocytometer. MATa cells (2×106) were mixed with MATα cells (1×107) in a final volume of 100 μL, incubated at 30°C for 5 hours, washed three times with water and resuspended in a final volume of 1 ml water. Then 20 μL of the cell suspension were plated on minimal media lacking lysine and tryptophan and containing histidine and leucine and incubated for 2 days at 30°C. This experiment was repeated at least three times and the mating efficiency was expressed as a percentage of diploid colonies formed by mating between the wild type MATa (LM102) and MATα (TBR1) strains. Reported values represent the mean of these tests. One-way ANOVA followed by Dunnett’s multiple comparison test test was used to determine statistical significance using GraphPad Prism (n = 3; p < 0.05).
To examine the role of the N-terminus of the yeast α-factor receptor Ste2p in receptor expression, we selected the first 30 residues in the N-terminus (See Fig. 1A for Ste2p snake diagram) based on several reasons including the presence of the two known glycosylation sites , the physical association of this region during mating , receptor dimerization [26, 27], the presence of several highly conserved residues, the pattern of solvent-accessibility , the possible interaction of this region with Ste3p , and intramolecular interaction with extracellular loop one . We carried out deletion mutagenesis of the N-terminal region generating five mutants [Ste2pΔ2-10, Ste2pΔ11-20, Ste2pΔ2-20, Ste2pΔ21-30, and Ste2pΔ2-30 (Fig. 1B)] in a background of the full-length, Cys-less receptor (Ste2p-C52S and C259S) with a His and FLAG tag extending from the C-terminus of Ste2p. (Fig. 1A). It has been established in previous studies in our lab and others that a single-Cys or a Cys-less “wild type” receptor is equivalent in activity to the naturally occurring wild type receptor [27, 32, 41–43]. All constructs presented in this study were based on this Cys-less, FLAG and HIS-tagged Ste2p construct that we refer to herein as the wild type (WT) receptor. All mutants as well as the wild type receptor were expressed from a high copy yeast expression vector under the control of the constitutive GPD promoter [9, 32].
To test the expression level of the mutant receptors, total membranes were prepared from yeast carrying each of the mutant constructs and the wild type control, the membrane proteins were solubilized and run on SDS-PAGE, immunoblotted, and probed with FLAG™ antibody (Fig. 2). As a loading control for immunoblot experiments, the same immunoblots were washed and re-probed with antibody against the constitutively expressed membrane protein Pma1p (Fig. 2, bottom panel). The experiment was repeated at least three times with similar results obtained in each experiment. Wild type Ste2p (Lane 2) appeared as a set of three major bands around 55 kDa, plus small amounts of higher molecular weight dimers (~100 kDa) and oligomers, as observed in many previous studies, for example [32, 45, 46]. Based on the primary amino acid sequence of the FLAG- and His-tagged receptor construct used in this study, the predicted molecular mass is 50.8 kDa . Glycosylation of the protein has been shown to result in the multiple molecular weights for the Ste2p monomer [25, 32, 47, 48]. The total expression levels as determined from total membrane preparation using FLAG-reactive bands of each mutant receptor in the total membrane preparation were found to range from 54% (Ste2pΔ2-30) to 81% (Ste2pΔ11-20) of the wild type (Table 1).
In addition to the reduced levels of expression, the mutants Ste2pΔ2-20, Ste2pΔ2-30, Ste2pΔ11-20, and Ste2pΔ21-30 exhibited altered banding patterns as compared with the wild type (Fig. 2; Fig. 3 lanes designated by C). We postulated that the different banding patterns were due to differential glycosylation of the mutant receptors as the two glycosylation sites of the receptor (N25 and N32) are located either within or adjacent to the deletion sites. To test this assumption, membranes were prepared from the mutants as well as the wild type cells, treated with PNGase F at 37°C to deglycosylate the receptor, and the banding pattern was examined. The higher molecular weight forms (greater than 130Kda) of Ste2p are attributable to receptor aggregation as a result of the incubation of the sample at 37°C for 2 hours which are conditions necessary for the deglycosylation reaction [Fig 3, Lanes (−) and Lanes (+)]. The aggregation of the receptor is likely responsible for the significant reduction in the overall intensity of the Ste2p monomer band after the treatment (Compare lanes C and − or + in Fig. 3). Such temperature-dependent aggregation has been observed previously by our lab . The results indicated that the variability in the number of bands was diminished after PNGase F treatment with all receptors exhibiting a prominent single band and the aforementioned high MW aggregate near the top of the gel (Fig. 3.). The collapse of the multiple bands at around 55 kDa into one major band at about 50 kDa after PNGase F treatment has been reported previously [25, 32]. Thus we conclude that the altered banding pattern in the mutants was due to differential glycosylation of the mutant receptors.
The immunoblot analysis measured receptor expression in the total membrane fraction (plasma membrane and intracellular membranes), but it did not provide information regarding receptor expression on the cell surface. Consequently, the surface expression of the mutants was determined by whole cell saturation binding assays using [3H]α-factor as described previously [32, 37, 41, 42]. The Bmax values from the saturation binding curves (Fig. 4) were used to calculate the number of receptors on the cell surface . The surface expression level of Ste2pΔ2-10 was approximately 90% of the wild type, but the surface expression levels of the other mutants were significantly lower (p<0.05) and varied between ~9% (Ste2pΔ2-30) and ~31% (Ste2pΔ2-20) of the wild type (Table 1). However, the binding affinity of the receptors as determined from the whole cell saturation binding assays indicates that the ligand-binding affinities (Kd values) of the mutant receptors were not significantly different (p>0.05) from that of the wild type receptor (Table 1). Thus we found that the N-terminal mutants are well expressed as judged by total expression levels but their surface expression was significantly reduced with the exception of Ste2pΔ2-10. Interestingly, the N-terminus had been reported earlier to be required for membrane orientation  and surface expression [27, 50]. The possibility exists that the decrease in receptor number at the cell surface is the result of an increased rate of receptor turnover. Finally our results pinpoint residues 11-30 of the N-terminus as required for optimal surface expression of Ste2p.
To investigate the role of the N-terminus in signaling activities, we examined the mutant receptors by pheromone-induced growth arrest and FUS1-lacZ induction assays. The growth arrest assay measures the cessation of growth of cells expressing Ste2p at the G1 phase based on the size of halos surrounding disks impregnated with various amounts of pheromone applied to lawn of pheromone-responsive cells. This assay measures response over a 24 to 48 h time frame. The FUS1-lacZ induction assay measures an early response (1 to 2 h) of the yeast cells to pheromone as detected by induction of β-galactosidase activity through a reporter gene construct consisting of a fusion between FUS1p, a pheromone-responsive promoter, and the lacZ gene . The signaling responses of the mutants in each experiment were normalized to those of the wild type.
In growth arrest assays, the halo diameter produced by the same amount of ligand was compared (Fig. 5, Table 2). The results showed that the halo sizes produced by the mutants were nearly the same as those of the wild type, with the exception of Ste2pΔ21-30 and Ste2pΔ2-30 which produced turbid halos (Fig. 5) which might be due to incomplete receptor activation or specific activation of a recovery pathway [30, 52]. Because either of these events can lead to reduced growth arrest, we conclude that residues 21-30 are required for efficient signaling.
Since the turbid halos did not start out as clear zones of growth inhibition that then filled in, which would have been an indication of recovery from growth arrest, the fact that the halos were never clear but turbid indicated that the Ste2pΔ21-30 and Ste2pΔ2-30 mutants did not undergo normal growth arrest. To further examine if the deletion mutants were compromised with respect to receptor activation, a liquid growth arrest assay was performed (Supplemental Material S1). Freshly sub-cultured cells were grown for four hours, then supplemented with α-factor (1 μM) and grown with shaking at 30C in a microtiter plate; cell density (OD600) was monitored at 15 minute intervals over the course of 12 hours. Duplicate cultures were treated in the same manner, but were not supplemented with pheromone. As expected, cells expressing the wild type receptor exhibited slowed growth in the presence of pheromone, while cells lacking the receptor continued to increase in numbers. Cells expressing the Ste2p 2-10 deletion were rapidly and completely growth arrested in the presence of pheromone, suggesting a heightened sensitivity to α-factor compared to the wild-type, reflecting the enhanced FUS1-lacZ activity (Fig. 6). Likewise, cells expressing the Ste2p 2-20 mutant had reduced growth in the presence of pheromone; these same cells had reduced signaling in the FUS1-lacZ assay (Fig. 6) but generated clear halos. In contrast, the Ste2p 2-30 deletion mutant, which had minimal FUS1-lacZ signaling activity (Fig. 6) and turbid halos (Fig. 5), did not exhibit any growth arrest in response to α-factor. These results support the hypothesis that receptor activation is compromised in the Ste2p 2-30 deletion mutant; this mutant exhibits high affinity binding (Table 1) but no evidence for activation is observed in the halo, FUS1-lacZ or growth arrest assays.
In FUS1-lacZ induction assays (Table 2 and Fig. 6), the maximal signaling activity, (FUS1-lacZ expression obtained at 1×10−6 M α-factor), varied among the receptors with signaling between 28% (Ste2pΔ2-30) and 152% (Ste2pΔ2-10) of the wild type (Table 2 and Fig. 6). Out of the five mutants tested, Ste2pΔ2-20, Ste2pΔ21-30, and Ste2pΔ2-30 exhibited a significant decrease in signaling (p<0.05), Ste2pΔ11-20 exhibited similar activity as the wild type and Ste2pΔ2-10 appeared to increase the signaling activity. Thus the signaling activities of the mutants as determined by three independent methods (halo assay, liquid growth arrest and FUS1-lacZ assays) indicated that the N-terminal residues 21-30 of Ste2p, play critical roles in signaling.
In the mating assay, the efficiency of Ste2pΔ2-20, Ste2pΔ21-30 and Ste2pΔ2-30 was similar to that of the wild type (Fig. 7). The other two mutants (Ste2pΔ2-10, Ste2pΔ11-20) exhibited statistically significant (p<0.05) enhanced mating efficiency. The mating efficiency of Ste2pΔ2-20 and Ste2pΔ2-30 reported in a previous study  was quite different from ours. In their experiment, although Ste2pΔ2-20 exhibited weak mating efficiency, the Ste2pΔ2-30 was completely sterile. We attribute the discrepancies in the mating efficiencies to the differences in receptor constructs. In the Shi et al. studies, Ste2p was fused to GFP whereas in our studies only the small epitopes (FLAG and His6) were fused to the C-terminus. Our experiments using Ste2p-tagged GFP revealed that GFP fusion, but not the epitope tags, affected both receptor trafficking and signaling. While growth arrest activity remained unchanged, appending the GFP tag onto WT Ste2p resulted in decreased FUS1-lacZ signaling. Both WT and truncated (Ste2p 2-10, Ste2p 2-20, Ste2p 2-30) GFP constructs were not localized to the plasma membrane (data not shown).
This study examined the role of the extracellular N-terminus in expression, signaling function and ligand-binding properties of yeast α-factor pheromone receptor Ste2p. On the basis of our findings, we propose that N-terminal residues 11-30 play a critical role in receptor cell surface expression. We also propose that residues 2-10 are involved in negative regulation of signaling based on the observation that deletion of these residues enhances mating and reporter gene induction. Furthermore, our results indicate that the residues 21-30 are essential for optimal signaling. Overall, our results suggest that the N-terminus of Ste2p plays multiple regulatory roles in controlling receptor function.
We found that deletion of residues 11-30 resulted in decreased cell surface expression. Similar results were observed with the N-terminal region of the dopamine D2 receptor and the neuropeptide Y subfamily receptors whereby truncation decreased cell surface expression of these receptors [53, 54]. However, N-terminal truncations do not always result in poor plasma membrane localization. Notably, truncation of the N-terminal region of the α1D-adrenoceptor (α1D-AR), the cannabinoid CB1 receptor, and GPR37 led to increased expression on the plasma membrane [18–20, 55]. All of these truncation studies of mammalian receptors were carried out in heterologous cell expression systems which might exert an influence receptor expression.
In this report, we studied expression of Ste2p in intact yeast cells, and found that truncation of the N-terminus led to lower Bmax values relative to wild type receptor, reflecting reduced receptors numbers on the cell surface as measured by whole cell binding assays. Interestingly, the N-terminal mutations did not affect ligand binding affinity (Kd), consistent with the observation by Sen and Marsh that this domain is not involved in ligand specificity . The decrease in Bmax for the mutant receptors suggested that N-terminal truncation either decreased receptor expression, decreased trafficking to the membrane or increased receptor degradation. Western blots analysis suggested that total cellular membrane receptor expression level of the mutants was only 1 to 2-fold lower than that of the wild type, despite up to 10-fold fewer radioligand binding sites on the cell surface (Table 1). This suggests that only a fraction of the Ste2p mutants detected in Western blots actually reached the cell surface. Therefore, our results indicate that shortening of the N-terminus is associated with reduced trafficking to the cell surface. Consistent with our observation are previous studies by Harley and Tipper who reported that the first 79 residues of the N-terminus of Ste2p are required for correct insertion and orientation of Ste2p into the plasma membrane .
All constructs expressed total receptor protein at similar levels, but the banding pattern of the mutants on SDS-PAGE was different from that of the wild type (Fig. 2 & 3). The changes in the banding pattern were due to altered glycosylation of the mutants likely owing to deletion of residues within, or proximity to, the glycosylation sites at N25 and N32 [25, 32]. Removal of the two glycosylation sites does not affect receptor activity or subcellular localization , thus reduced surface expression cannot be attributed simply to changes in the glycosylation state. It has been reported that the charged residues in the N-terminus are important for proper orientation of the receptor in the membrane . It is reasonable that a combination of the modification in the glycosylation state coupled with changes in the electrostatic properties of the N-terminus influence the localization of Ste2p and its orientation in the plasma membrane.
Deletion of residues within the N-terminus affected signaling activities of the receptor as examined by pheromone-induced growth arrest assay of the mutants. Three mutants (Ste2pΔ2-10, Ste2pΔ11-20, and Ste2pΔ2-20) exhibited wild type growth arrest. Two other mutants (Ste2pΔ21-30 and Ste2pΔ2-30) exhibited significantly reduced signaling response (Table 2) yielding very turbid halos with indistinct edges. This observation is consistent with previous studies in which deletion of residues 2-30 produced small, fuzzy halos  and this halo phenotype has been observed by others for a variety of different Ste2p mutations [52, 57, 58]. It has been shown that a small number of receptors on the cell surface is sufficient to yield a clear, not fuzzy, halo with small diameter [59–61]. We conclude that the fuzzy halos produced by the N-terminal deletion mutants that bind ligand efficiently are not due to low receptor number on the cell surface. We assume that a conformational change required for full activation of the receptor does not occur in the mutants; signaling response is poor resulting in a fuzzy halo. Alternately, a fuzzy halo may result from enhanced rates of recovery from pheromone stimulation. Feedback mechanisms have evolved to restrict the duration of signaling and stimulate recovery from pheromone-induced G1 arrest. Several mechanisms have evolved for this purpose including downregulation of Ste2p by endocytosis, upregulation of the expression of an α-factor-degrading protease (Bar1), and stimulation of Gpa1-GTP hydrolysis by Sst2.
Deletion of residues 2-10 (Ste2p 2-10) resulted in enhanced signaling (~150% relative to WT) as assessed in the FUS1-lacZ assay, suggesting that the presence of this region of the N-terminus normally serves to inhibit receptor activity. Enhanced signaling has been noted for other GPCR with N-terminal deletions, including GPR56, an adhesion GPCR  and the serotonin 5-HT2B receptor . Truncation of the brain-specific receptors angiogenesis inhibitor-1 (BAI1)  and the orphan GPR61 receptor  resulted in constitutive activity, suggesting that the N-terminus constrains activity of these receptors. In contrast, deletion of residues 2-20 (Ste2pΔ2-20) resulted in a receptor with compromised signaling activity (~25% of WT), indicating that while deletion of 2-10 should enhance activity, the net effect of the combined deletion (2-10 plus 11-20) reduced receptor activity below that of the WT. Interestingly, deletion of residues 11-20 (Ste2p 11-20) yielded a receptor with near WT activity, a phenotype intermediate to the Ste2p 2-10 (enhanced signaling) and Ste2p 2-20 (reduced signaling) receptors. In the absence of the 11-20 residues, the N-terminus likely assumes a different conformation. It is possible that the 11-20 deletion of the N-terminus may ameliorate the inhibitory influence of the 2-10 residues, with the net effect being signaling at a WT level. This hypothesis will warrant further study.
As noted above, for some mutants there was discordance in the functional readouts of the growth arrest and signaling assays. Such divergence in correlation of the response measured for the assessments of receptor activation has been observed in many studies. For example, the Ste2p mutantsY266C, Q149R and W295C exhibited robust FUS1-lacZ activity, but little to no growth arrest activity   . Similarly, a divergence in correlation between mating efficiency and FUS1-lacZ induction has been reported for I24C and I29 mutants which exhibited approximately 50% and 100% of the WT FUS1-lacZ activities, respectively, but only <0.09% and 4.6% of the WT mating efficiency. Mutations of receptors resulting in biased activation of specific signaling pathways has been described for other GPCRs [67–70].
The involvement of the N-terminus in negative regulation of Ste2p suggests that this domain may play a role in the transition from the inactive to the active state of Ste2p. The deletion of residues 2-10 does not lead to constitutively activity indicating that the N-terminus is not required to stabilize the inactive receptor conformation. However, Ste2p activation by pheromone is up-regulated when the 2-10 residues are truncated, suggesting that the N-terminus influences the conformational state of other domains of that are important for signal transduction. The N-terminus, in particular residues 20-30, is predicted to form a β-sheet, [27, 28]. Removal of these residues has the potential to reduce β-sheet like contacts within Ste2p or even with domains of the Ste2p dimer [26, 27], which could promote the transition to an activated state upon ligand binding. We speculate that extracellular loop one (EL1), which is predicted to exist as a 3-10 helix, might interact with the N-terminus, as mutations in this loop change the glycosylation patterns of the receptor and influences its ability to be activated. [28, 32]. However, we do not exclude the possibility of interactions with other Ste2p domains.
In conclusion, our results and those from mammalian GPCRs indicate an increasing importance for the N-terminus in the biology of GPCRs. Although the underlying interactions between receptor domains/residues within the receptor, between receptor oligomers, or between the receptor and other membrane proteins remain unknown, the deletions studied herein may allow new, unnatural interactions that affect function. The N-terminus may be required for fine tuning the signaling process and thus may provide an important future drug target to regulate GPCR function.
We thank Li-Yin Huang for help in the binding assays.
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