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. 2023 Aug 1;34(9):br14. doi: 10.1091/mbc.E23-03-0117

Serotonin 5-HT7 receptor slows down the Gs protein: a single molecule perspective

Aleš Petelák a, Nevin A Lambert b, Alexey Bondar c,d,*
Editor: JoAnn Trejoe
PMCID: PMC10398887  PMID: 37342875

Abstract

The 5-hydroxytryptamine (serotonin) receptor type 7 (5-HT7R) is a G protein–coupled receptor present primarily in the nervous system and gastrointestinal tract, where it regulates mood, cognition, digestion, and vasoconstriction. 5-HT7R has previously been shown to bind to its cognate stimulatory Gs protein in the inactive state. This phenomenon, termed “inverse coupling,” is thought to counteract the atypically high intrinsic activity of 5-HT7R. However, it is not clear how active and inactive 5-HT7 receptors affect the mobility of the Gs protein in the plasma membrane. Here, we used single-molecule imaging of the Gs protein and 5-HT7R to evaluate Gs mobility in the membrane in the presence of 5-HT7R and its mutants. We show that expression of 5-HT7R dramatically reduces the diffusion rate of Gs. Expression of the constitutively active mutant 5-HT7R (L173A) is less effective at slowing Gs diffusion presumably due to the reduced ability to form long-lasting inactive complexes. An inactive 5-HT7R (N380K) mutant slows down Gs to the same extent as the wild-type receptor. We conclude that inactive 5-HT7R profoundly affects Gs mobility, which could lead to Gs redistribution in the plasma membrane and alter its availability to other G protein–coupled receptors and effectors.

INTRODUCTION

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that plays a profound role in the function of the nervous system, gastrointestinal tract, and cardiovascular system (Berger et al., 2009). The multifaceted action of 5-HT regulates mood, sleep, cognition, memory, appetite, digestion, body temperature, and vasoconstriction (Guzel and Mirowska-Guzel, 2022). Malfunctions of 5-HT signaling can lead to depression, anxiety, migraine, irritable bowel syndrome, and obsessive–compulsive disorder (Pourhamzeh et al., 2022).

The 5-HT receptor type 7 (5-HT7R) is a G protein–coupled receptor (GPCR) that belongs to the serotonin receptor family and plays a role in several physiological processes, including the regulation of mood, sleep, and cognitive function (Guseva et al., 2014). 5-HT7R may be involved in the mechanism of action of antidepressant medications (Hedlund, 2009) and has been studied as a potential target for the treatment of a variety of psychiatric conditions, including depression, anxiety, and insomnia (Gasbarri and Pompili, 2014), as well as intestinal disorders (Kim and Khan, 2014).

Unlike most GPCRs, 5-HT7R has been shown to bind to its cognate stimulatory G protein (Gs) in the inactive state (Andressen et al., 2018; Ulsund et al., 2019; Jang et al., 2020). This interaction, termed “inverse coupling,” is likely required to counteract the outstandingly high basal activity of 5-HT7R (Jang et al., 2020). Therefore, 5-HT7R stimulation with 5-HT has a twofold effect because it induces the binding of Gs molecules to the activated 5-HT7R, as well as activation and dissociation of preassembled 5-HT7R/Gs complexes. However, the effect of inverse coupling on the localization and mobility of Gs remains unclear. Here, we used single-molecule imaging of the Gs protein and 5-HT7R in living cells to determine the effect of 5-HT7R and its mutants on the mobility of Gs molecules.

RESULTS AND DISCUSSION

We first determined the diffusion coefficients of Gs (when 5-HT7R was not present) and 5-HT7R (Figure 1). In experiments with Gs, we utilized the Gγ2 subunit N-terminally labeled with HaloTag (Halo-Gγ2) (Los et al., 2008) as described previously (Bondar et al., 2020), expressed at a low level of 0.28 ± 0.19 molecules/μm2 (mean ± SD) suitable for single-molecule imaging (<1 molecule/μm2) under control of a minimal promoter (minP; Figure 1, A and B). We used the labeled Gγ2 subunit to avoid alterations that often accompany the labeling of Gα subunits of G proteins (Bondar and Lazar, 2014). To ensure the preferential assembly of the labeled Halo-Gγ2 in Gs heterotrimers, we also overexpressed nonlabeled Gαs and Gβ1 subunits. G proteins were then fully labeled with the Halo-JF646 dye. Importantly, Chinese hamster ovary cells (CHO-K1) that were used in experiments do not endogenously express 5-HT7R (Mahe et al., 2004). The 5-HT7 receptor was labeled with the SNAPf-tag (Keppler et al., 2003) at its N-terminus (SNAPf-5-HT7R) and overexpressed in CHO-K1 cells. For single-molecule imaging of SNAPf-5-HT7R, we underlabeled a small fraction of the total 5-HT7R pool with the SNAP-Surface549 dye (Figure 1, A and C). Single-molecule tracking showed profoundly higher mobility of Gs (observed by imaging Halo-Gγ2 in presence of overexpressed Gαs and Gβ1) (Figure 1, D and E) in the membrane compared with SNAPf-5-HT7R (Figure 1E). Initial observed trajectories were segmented into individual tracks using DC-MSS software (Vega et al., 2018) based on the motion type (immobile, confined, free, and directed) exhibited by the segments. The median diffusion coefficient of Gs was 0.292 μm2/s with 53% of molecules exhibiting free diffusion motion type (Figure 1F). In contrast, SNAPf-5-HT7R had a median diffusion coefficient of 0.021 μm2/s with a large fraction of immobile molecules (37%) (Figure 1F). Negligible nonspecific staining was observed with the SNAP-Surface549 dye (200 pM) in control experiments. The median diffusion coefficients obtained from individual tracks (Figure 1G) were consistent with those obtained from individual cells (Figure 1F). The distribution of diffusion coefficients of individual tracks confirmed a higher abundance of low-mobility tracks of SNAPf-5-HT7R molecules compared with Gs (Figure 1G). These findings show that the Gs protein shows almost 10 times higher mobility than 5-HT7R (P < 0.0001), as expected for membrane-associated and transmembrane proteins, respectively. The striking differences in mobility between these peripheral and integral membrane proteins suggest that the presence of long-lasting complexes between these signaling partners will have a profound effect on Gs mobility.

FIGURE 1:

FIGURE 1:

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Single-molecule imaging of Gs and 5-HT7R mobility. (A) Experimental design and labeling strategy. The N-terminus of 5-HT7R was tagged with the SNAP-tag and labeled with the SNAP-Surface549 dye. The Gs protein was tagged at the N-terminus of the Gγ2 subunit with the HaloTag and labeled with the Halo-JF646 dye. (B) A CHO-K1 cell with low (0.28 ± 0.19 molecules/μm2 [mean ± SD]) expression of Halo-Gγ2 fully labeled with the Halo-JF646 dye (50-nM final concentration). (C) A CHO-K1 cell overexpressing SNAPf-5-HT7R underlabeled with the SNAP-Surface549 dye (200-pM final concentration). Scale bar in B and C 10 μm. (D) Tracks of the Halo-Gγ2 movement obtained by observation of a cell region for 5 s in the presence of cotransfected Gαs and Gβ1 and in the absence of 5-HT7R. (E) Tracks of the SNAPf-5-HT7R movement obtained by observation of a cell region for 5 s in the absence of cotransfected Gs subunits. Scale bar in D and E: 1 μm. The color coding of the tracks in D and E indicates the molecule displacement (as indicated by the color bar). (F) Median diffusion coefficients with 95% confidence interval of individual cells expressing labeled Gs (magenta; n = 34, m = 3) or 5-HT7R (gray; n = 27, m = 6). The receptor demonstrates significantly lower mobility than Gs (P < 0.0001, Mann–Whitney test). (G) Comparison of the distribution of diffusion coefficients of individual tracks of labeled Gs (magenta, 10,527 tracks) or 5-HT7R (gray, 5124 tracks). n : number of analyzed individual cells. m : number of independent experiments.

Next, we set out to determine how 5-HT7R and its inactive and constitutively active mutants affect Gs mobility (Figure 2). We used the N-terminally SNAPf-tagged wild-type 5-HT7R, its constitutively active mutant 5-HT7R (L173A), or the inactive mutant 5-HT7R(N380K) described previously (Jang et al., 2020). We co-transfected CHO-K1 cells with Gαs, Gβ1, and minP-Halo-Gγ2 subunits together with SNAPf-5-HT7R or its mutants and monitored Gs mobility. As a positive control, we also studied the mobility of HaloTag-labeled mini Gs (mGs), an engineered G protein surrogate that is expected to form long-lasting complexes with active Gs-coupled receptors (Nehme et al., 2017). Typical results of Gs single-molecule tracking are shown in Figure 2, A–C. The median diffusion coefficient of Gs in presence of SNAPf-5-HT7R was 0.112 μm2/s, indicating a significant (P < 0.0001) decrease in the overall mobility of Gs by 62% and the appearance of a large fraction of immobile molecules (Figure 2, A, F, and I). Diffusion coefficients of these immobile molecules (D < 0.009 μm2/s) likely indicated limits of detection precision rather than true mobility. The mobility of Gs was less affected in the presence of the active mutant SNAPf-5-HT7R(L173A) (median D = 0.181 μm2/s) (Figure 2, B, F, and J). Gs mobility in presence of SNAPf-5-HT7R(L173A) was significantly lower than without a coexpressed receptor (P = 0.0427) but higher than in presence of wild-type 5-HT7R (P = 0.0229). However, the expression of the inactive mutant SNAPf-5-HT7R(N380K) led to a reduction in Gs mobility (median D = 0.114 μm2/s) (Figure 2, C, F, and K) to a similar extent as the expression of the wild-type SNAPf-5-HT7R (median D = 0.112 μm2/s) (P > 0.9999). Both mutants of 5-HT7R showed mobility similar to that of the wild-type receptor (Figure 2, G and N). As expected, Halo-mGs that appeared in the plasma membrane in cells expressing SNAPf-5-HT7R (stimulated with 10-μM 5-HT) exhibited very low mobility (median diffusion coefficient 0.039 μm2/s; Figure 2, D, F, and L) that closely resembled that of the receptor (P > 0.9999) and was considerably lower than the diffusion coefficients observed for Gs (P < 0.0001). Expression of a prototypical Gs-coupled SNAPf-β2-adrenergic receptor (SNAPf-β2AR), which does not exhibit inverse coupling, did not have a significant effect on Gs mobility (Figure 2 E, H, and M) (P > 0.9999). The mobility differences obtained from median diffusion coefficients of individual cells (Figure 2F) were confirmed by distinct mobility distributions of individual tracks pooled from all cells in the tested experimental combinations (Figure 2, I–N). Gs tracks exhibited a broad distribution of diffusion coefficients, which consistently shifted toward lower mobility when SNAPf-5-HT7R variants were present.

FIGURE 2:

FIGURE 2:

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Inactive 5-HT7R slows down Gs mobility. (A–C) Tracks of single Halo-Gγ2 molecules cotransfected with Gαs and Gβ1 in presence of SNAPf-5-HT7R (A), constitutively active mutant SNAPf-5-HT7R(L173A) (B), and inactive mutant SNAPf-5-HT7R(N380K) (C). (D) Tracks of Halo-mGs molecules in the presence of SNAPf-5-HT7R stimulated with 10 μM 5-HT. (E) Tracks of single Halo-Gγ2 molecules cotransfected with Gαs and Gβ1 in presence of SNAPf-β2AR. Scale bar in A–E: 1 μm. The color coding of the tracks in A–E indicates the molecule displacement (as indicated by the color bar). (F) Median diffusion coefficients of Gs with 95% confidence interval in individual cells expressing Gs with SNAPf-5-HT7R (red; n = 40, m = 7), SNAPf-5-HT7R(L173A) (blue; n = 34, m = 3), SNAPf-5-HT7R(N380K) (yellow; n = 23, m = 3), and median diffusion coefficients of Halo-mGs in cells expressing SNAPf-5-HT7R and activated with 10-μM 5-HT (green; n = 18, m = 3). The median diffusion coefficients of Gs and SNAPf-5-HT7R expressed separately are indicated by dashed lines. Gs is significantly slower when coexpressed with SNAPf-5-HT7R (P < 0.0001), SNAPf-5-HT7R(L173A) (P = 0.0128), and SNAPf-5-HT7R(N380K) (P < 0.0001) than without a coexpressed GPCR. Similarly, membrane-localized mGs bound to the active SNAPf-5-HT7R is significantly slower (P < 0.0001) than Gs without a coexpressed receptor. (G) Median diffusion coefficients with 95% confidence interval of SNAPf-5-HT7R (red; n = 17, m = 5), SNAPf-5-HT7R(L173A) (blue; n = 21, m = 3), SNAPf-5-HT7R(N380K) (yellow; n = 9, m = 3), and activated SNAPf-5-HT7R (green; n = 17, m = 3) in individual cells. No significant differences in mobility of expressed receptors were found. (H) Median diffusion coefficients with 95% confidence interval of Gs (violet; n = 33, m = 6) and SNAPf-β2AR (gray; n = 20, m = 4) in individual cells expressing Gs in presence of SNAPf-β2AR. Gs mobility in presence of SNAPf-β2AR is not significantly different from that in the absence of a GPCR (P > 0.9999). (I–M) Comparisons of the diffusion coefficient distribution of individual tracks of Gs with SNAPf-5-HT7R (I; red: Gs [20,044 tracks]; black dashed line: 5-HT7R [3975 tracks]), constitutively active SNAPf-5-HT7R(L173A) (J; blue: Gs [11,966 tracks]; black dashed line: 5-HT7R(L173A) [6480 tracks]), inactive SNAPf-5-HT7R(N380K) (K, yellow: Gs [7566 tracks]; black dashed line: 5-HT7R(N380K) [3448 tracks]), Halo-mGs with activated SNAPf-5-HT7R (L, green: mGs [5893 tracks]; black dashed line: activated 5-HT7R [8542 tracks]), and SNAPf-β2AR (M, violet: Gs [16,715 tracks]; black dashed line: β2AR [2499 tracks]). (N) Comparisons of the diffusion coefficient distribution of individual tracks of Gs and all tested GPCRs and mutants. In I–N, the distribution of the diffusion coefficients for Gs expressed separately is indicated by the magenta dashed line. The expression of 5-HT7R strongly reduces the mobility of Gs (P < 0.0001). The presence of the constitutively active mutant SNAPf-5-HT7R(L173A) has a smaller effect on the mobility of Gs (P = 0.0128). Expression of the inactive mutant SNAPf-5-HT7R(N380K) leads to a reduction of the Gs mobility to a similar extent as the nonmutated 5-HT7R. The mobility of Halo-mGs localized in the membrane in the presence of SNAPf-5-HT7R is considerably lower than that of Gs (P < 0.0001) and comparable with the mobility of the receptor itself (P > 0.9999). Expression of the SNAPf-β2AR does not affect Gs mobility (P > 0.9999). n: number of analyzed individual cells; m: number of independent experiments. Statistical testing was done using multiple comparisons nonparametric Kruskal–Wallis test with Dunn’s correction.

Our findings indicate that the presence of wild-type or inactive mutant 5-HT7R significantly slows the mobility of Gs protein heterotrimers in the plasma membrane, consistent with previous ensemble measurements of Gs mobility (Andressen et al., 2018). This is most likely due to the formation of complexes between the inactive 5-HT7R and Gs that exhibit the low mobility of the receptor. That Gs mobility remains higher than 5-HT7R mobility under these conditions suggests that inactive-state complexes are transient (Jang et al., 2020), and there is a fraction of free Gs molecules even in cells overexpressing 5-HT7R. It is also possible that a small fraction of Halo-Gγ2 associates with endogenous Gα subunits of other subtypes. Inactive-state complexes do not form when 5-HT7R is present in its active state, but instead conventional active-state complexes would be expected. Slowing of Gs mobility by the constitutively active 5-HT7R(L173A) mutant can be explained by such active-state complexes and possibly by receptors that can sample the inactive state despite the activating mutation. It is also possible that binding of activated Gs subunits to slow-moving effectors and regulators could also reduce their mobility (Bondar et al., 2020). In either case, our results are consistent with the suggestion that inactive-state 5-HT7R-Gs complexes are more stable than active-state complexes (Jang et al., 2020). It remains unclear whether 5-HT7R forms inactive-state complexes with other G proteins, particularly with the G12 protein. Potential roles of membrane domains and endosomal compartments in regulation of abundance and lifetimes of inactive-state 5-HT7R-Gs complexes remain insufficiently understood. Overall, we conclude that the presence and the activation state of 5-HT7R have a major effect on the mobility of Gs. This in turn affects the availability of Gs to other binding partners and could affect its spatial distribution in the cellular plasma membrane. For example, clustering of 5-HT7R in certain compartments of the plasma membrane could cause a selective accumulation of Gs molecules in such areas and their inaccessibility to other partner proteins. Such effects would be expected to depend critically on the overall density of 5-HT7R in native cells and tissues.

MATERIALS AND METHODS

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DNA constructs

Halo-Gγ2 with expression controlled by the minimal promoter (minP, Promega) and SNAPf-β2AR were described previously (Bondar et al., 2020). Halo-mGs was created by replacing Venus in the Venus-mGs with HaloTag. The constructs SNAPf-5-HT7R, SNAPf-5-HT7R(L173A), and SNAPf-5-HT7R(N380K) were created by cloning the 5-HT7R sequence from the PRESTO-Tango GPCR kit (Kroeze et al., 2015) into the SNAPtag-C1 vector and subsequently mutating individual amino acids for the L173A and N380K mutants using Quikchange mutagenesis. Nontagged Gαs and Gβ1 subunits were purchased from cdna.org.

Cell culture

We performed the experiments in CHO-K1 cells (American Type Culture Collection). The cells were cultured at +37°C and 5% CO2 in the F-12K medium (ThermoFisher Scientific, Waltham, MA) supplemented with fetal bovine serum (Merck, Rahway, NJ) and antibiotic/antimycotic solution (Merck). The cells were passaged twice a week. Cells were plated on plastic six-well plates and cultivated at least 24 h prior to transfection. Transfection was done using PEI-Max (Polysciences, Warrington, PA).

Sample preparation

The preparation of cells for imaging is described in detail in Bondar et al. (2020). Briefly, cells were grown and transfected in regular six-well plastic plates. Then 24 h after transfection, cells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS) and labeled with Halo-JF646 (50-nM final concentration for full labeling) and SNAP-Surface549 (200 pM final concentration for underlabeling). Cells were incubated with the dyes for 30 min, washed three times with DPBS without Ca2+ and Mg2+ and dislodged using Accutase (Merck). Dislodged cells were spun three times in DPBS at 300 g for 4 min and plated on extra-clean glass coverslips coated with fibronectin (Merck). Cells were incubated on glass coverslips for 1 h before imaging, which was sufficient for efficient adhesion to the coverslip.

Single-molecule microscopy

Imaging of single molecules was done using Total Internal Reflection Fluorescence (TIRF) microscopy (Axelrod, 1981). We used the Olympus IX83 inverted microscope equipped with the TIRF1 illuminator, 561-nm and 640-nm lasers (100 mW each), 60 × 1.49NA UApoplan objective lens (Olympus), TRF89901-OL3 TIRF quad-band dichroic beamsplitter (Chroma), Zyla 4.2 sCMOS camera (Andor), and a sample heater (Stable Z, Bioptechs). The 652LP dichroic beamsplitter and the 682/40 emission filter were used for imaging at 640-nm excitation, and the 565LP dichroic beamsplitter and the 605/50 emission filter were used for imaging at 561-nm excitation. Imaging of individual cells was performed for 5 s at 33 fps for the 640-nm channel and 18 fps for the 561-nm channel using the 25-mW laser power. In all experiments involving 5-HT7R constructs, receptor presence was confirmed by imaging.

Data analysis

Detection of single particles and their tracking was performed using the TrackMate plugin (Tinevez et al., 2017) in Fiji using the LoG detector and the simple LAP tracker. The object diameter was estimated to be 0.4 µm. The quality threshold was manually adjusted for individual time series. The maximal linking distance between two frames was set to 0.5 µm. The median filter and subpixel localization were used. Initial trajectories of at least 20 frames were segmented into individual tracks based on their diffusion type. The diffusion coefficients for individual tracks and their diffusion type were determined using the DC-MSS software (Vega et al., 2018) in MATLAB using the default parameters.

Statistical analysis

The median diffusion coefficients with 95% confidence interval were obtained from individual cells and pooled for separate experimental conditions. Individual tracks from multiple cells were also pooled together for verification to produce a median diffusion coefficient based on all tracks. Normality testing was done using D’Agostino & Pearson omnibus k2 test. Individual datasets where appropriate were compared using two-tailed Mann–Whitney test. Statistical analysis and comparison of multiple experimental conditions were performed using the nonparametric Kruskal–Wallis test with Dunn’s comparison of individual conditions.

Supplementary Material

Click here for additional data file. (164KB, pdf)

Acknowledgments

The authors thank Prof Luke Lavis (HHMI, Janelia Research Campus) for providing the Halo-JF646 dye. This work was supported by National Institutes of Health (NIH) Grants GM130142 and GM145284 (to N.A.L.), the Czech Science Foundation Grant 20-09628Y, and the Czech Ministry of Education, Youth, and Sports Grant Inter-COST LTC20074 (to A.B.).

Abbreviations used:

5-HT

5-hydroxytryptamine (serotonin)

5-HT 7R

5-hydroxytryptamine (serotonin) receptor type 7

CHO-K1

Chinese hamster ovary cells

GPCR

G protein–coupled receptor

G s

stimulatory G protein

Halo

HaloTag

mG s

miniG stimulatory protein

SNAPf

SNAP-tag.

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E23-03-0117) on June 21, 2023.

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Associated Data

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Supplementary Materials

Click here for additional data file. (164KB, pdf)

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