Homodimer Biogenesis

There are several lines of evidence suggesting that GPCR dimerization occurs prior to receptor expression on the plasma membrane. The most notable example involves heterodimerization of class C GABAbR1 and GABAbR2 receptors, which has been demonstrated to be essential for receptor trafficking from the ER to the plasma membrane (Jones et al. 1998; Kaupmann et al. 1998; White et al. 1998;

Margeta-Mitrovic et al. 2000). Dimerization has been proposed as a general mechanism necessary for proper trafficking of class A GPCR to the plasma membrane (Terrillon and Bouvier 2004). Experimental evidence in favor of this model includes the identification of constitutive homodimers on the plasma membrane (HerrickDavis et al. 2004; McVey et al. 2001), the identification of nontrafficking, mutant receptors that dimerize with and retain their wild-type counterparts within intracel-lular compartments (Benkirane et al. 1997; Zhu and Wess 1998; Karpa et al. 2000; Shioda et al. 2001; O'Dowd et al. 2005) and the detection of positive BRET signals in both plasma membrane and endomembrane-enriched sub-cellular fractions prepared from HEK 293 cells expressing vasopressin or b2-adrenergic receptors (Terrillon et al. 2003; Salahpour et al. 2004). While these results suggest that GPCR dimerization may occur within intracellular compartments, direct evidence demonstrating the formation of class A GPCR homodimers within the ER and Golgi of intact living cells was not provided by these studies.

To address the issue of 5-HT2C receptor homodimer biogenesis, confocal microscopy combined with FRET was used to measure protein-protein proximity within the ER and Golgi of living cells (Herrick-Davis et al. 2006). The confocal microscopy-based FRET approach allows visualization of specific intracellular compartments, without compromising cellular integrity, and allows real-time monitoring of GPCR dimerization in the ER and Golgi apparatus. To begin this study, live-cell confocal fluorescence imaging was first performed on HEK 293 cells expressing an ER-YFP marker (a mutant YFP protein with an ER targeting and retention sequence) and Golgi-YFP marker (YFP containing a Golgi targeting sequence) to establish direct visualization of ER and Golgi membranes. ER membranes appear as a diffuse reticular network spreading outward from the nucleus to the plasma membrane (Fig. 7.5a). In contrast, Golgi membranes are visualized as dense punctate areas of fluorescence in the perinuclear region (Fig. 7.5b). A membrane impermeable dye, Dil, labels the plasma membrane (Fig. 7.5c).

Real-time imaging of CFP- and YFP-tagged 5-HT2C receptors was performed 12-24 h posttransfection to visualize 5-HT2C receptors following biosynthesis in the ER, trafficking through the Golgi, and subsequent expression on the plasma membrane (Herrick-Davis et al. 2006). In cells cotransfected with 5-HT2C/CFP and the ER-YFP marker, CFP and YFP were colocalized within the ER 12 h posttrans-fection. In cells cotransfected with 5-HT2C/CFP and the Golgi-YFP marker, CFP and YFP were colocalized within the Golgi 16 h posttransfection. By 20 h post-transfection, 5-HT2C/CFP fluorescence was on the plasma membrane, while the Golgi-YFP marker remained in the Golgi (Herrick-Davis et al. 2006). Based on these results, a similar time course was performed in cells cotransfected with 5-HT2C/CFP and 5-HT2C/YFP. The cells were imaged live using confocal fluorescence imaging at various time points posttransfection. Ten hours after the addition of transfection reagents to HEK 293 cells, 5-HT2C/CFP and 5-HT2C/YFP receptor fluorescence began to emerge. Twelve hours posttransfection, confocal fluorescence imaging revealed many cells with a diffuse reticular pattern of fluorescence, similar to pattern of fluorescence observed for the ER-YFP marker (Fig. 7.5d). Sixteen hours posttransfection, most of the cells displayed a more clustered and

5-HT2C

v pi

12hr 16hr 20hr

Time posttransfection

Fig. 7.5 Confocal microscopy of HEK 293 cells expressing markers for different cellular compartments (a)-(c) and at various time points posttransfection with 5-HT2C receptors (d)-(f). (a) HEK cell expressing the ER/YFP marker. (b) HEK cell expressing the Golgi/YFP marker. (c) DiI labeling of plasma membrane. (d) 5-HT2C/YFP 12 h posttransfection. (e) 5-HT2C/YFP 16 h posttransfection. (f) 5-HT2C/YFP 20 h posttransfection. Scale bar = 10 mm dense pattern of labeling with a distinct perinuclear distribution, in a manner very similar to the fluorescence from the Golgi-YFP marker (Fig. 7.5e). By 20 h post-transfection, most of the cells were expressing 5-HT2C receptors on the plasma membrane (Fig. 7.5f).

Confocal microscopy and acceptor photobleaching FRET were performed within discrete regions of the ER and Golgi, 12 and 16 h posttransfection, of HEK 293 cells coexpressing 5-HT2C/CFP and 5-HT2C/YFP (Herrick-Davis et al. 2006). Laser scanning confocal microscopy allows the photobleaching to be confined to very discrete intracellular regions. This minimizes the time required for irradiation of the acceptor fluorophore and makes the technique suitable for live-cell imaging. Acceptor photobleaching FRET experiments were performed in 50 living HEK 293 cells coexpressing 5-HT2C/CFP and 5-HT2C/YFP in the ER (12 h posttransfection) and in 50 cells coexpressing 5-HT2C/CFP and 5-HT2C/YFP in the Golgi (16 h post-transfection). The results are shown in Fig. 7.6a and b. A specific FRET signal resulting from proteins in a clustered distribution, such as dimers/oligomers, has

Fig. 7.6 Acceptor photobleaching FRET was measured in the ER and Golgi of live HEK 293 cells expressing CFP- and YFP-tagged 5-HT2C receptors. FRET efficiency was calculated as 100 x [(CFP postbleach - CFP prebleach)/CFP postbleach] using Zeiss Aim Software. (a) FRET efficiency was plotted versus uD/A ratio (postbleach CFP fluorescence/prebleach YFP fluorescence) for cells coexpressing 5-HT2C/CFP and 5-HT2C/YFP in the ER (n = 50) and in the Golgi (n = 50). Nonlinear regression analysis (one-phase exponential decay, GraphPad Prism) yielded R2 = 0.91. (b) FRET efficiency plotted versus YFP fluorescence for the same 100 cells as shown in Fig. 7.7a. Linear regression analysis (GraphPad Prism) revealed no correlation between FRET efficiency and YFP fluorescence levels (r2 < 0.1)

Fig. 7.6 Acceptor photobleaching FRET was measured in the ER and Golgi of live HEK 293 cells expressing CFP- and YFP-tagged 5-HT2C receptors. FRET efficiency was calculated as 100 x [(CFP postbleach - CFP prebleach)/CFP postbleach] using Zeiss Aim Software. (a) FRET efficiency was plotted versus uD/A ratio (postbleach CFP fluorescence/prebleach YFP fluorescence) for cells coexpressing 5-HT2C/CFP and 5-HT2C/YFP in the ER (n = 50) and in the Golgi (n = 50). Nonlinear regression analysis (one-phase exponential decay, GraphPad Prism) yielded R2 = 0.91. (b) FRET efficiency plotted versus YFP fluorescence for the same 100 cells as shown in Fig. 7.7a. Linear regression analysis (GraphPad Prism) revealed no correlation between FRET efficiency and YFP fluorescence levels (r2 < 0.1)

been shown to be dependent on the ratio of donor to acceptor expressed in the cell, while FRET resulting from overexpression of randomly distributed proteins can be distinguished by a dependence on acceptor fluorescence or receptor expression level (Kenworthy and Edidin 1998; Wallrabe et al. 2003). Real-time FRET efficiencies measured for 5-HT2C/CFP and 5-HT2C/YFP fusion proteins in the ER and Golgi apparatus were dependent on the ratio of donor to acceptor (uD/A) expressed within a given cell (Fig. 7.6a) and independent of receptor expression level, measured as acceptor or YFP fluorescence (Fig. 7.6b). FRET efficiencies for a given uD/A ratio were similar for ER, Golgi, and plasma membrane. The mean FRET efficiency was 21.4% for ER, 20.3% for Golgi, and 21.5% for plasma membrane. These results suggest that 5-HT2C receptor homodimerization begins during receptor biosynthesis within the ER and is a naturally occurring step in 5-HT2C receptor maturation and processing (Herrick-Davis et al. 2006).

7.4 Functional Significance of 5-HT2C Receptor Dimerization: Determining the Ligand/Dimer/G-Protein Stoichiometry

Homodimerization and heterodimerization between different families of GPCR have been reported to regulate ligand binding, second messenger activation, and receptor trafficking (reviewed in Angers et al. 2002), (Javitch 2004), and (Milligan 2004).

While these studies suggest that dimerization may be a common property of GPCR, the specific mechanisms and functional consequences of dimerization may differ from one GPCR to another. For GABAB receptors the functional significance of dimerization is clear: heterodimerization of GABAbR1 and GABAbR2 is essential for proper plasma membrane targeting, ligand binding and signal transduction (Jones et al. 1998; Kaupmann et al. 1998; White et al. 1998; Margeta-Mitrovic et al. 2000). However, for many GPCR the functional significance of dimerization remains unknown. To address this issue, studies were performed to determine if dimerization plays a functional role in regulating the activity of 5-HT2C receptors. Radioligand binding, inositol phosphate (IP) signaling, confocal imaging and FRET were evaluated in HEK 293 cells coexpressing wild-type 5-HT2C receptors along with an inactive, mutant 5-HT2C receptor to determine the effect of dimeriza-tion on receptor function and to investigate the ligand/dimer/G-protein stoichiom-etry (Herrick-Davis et al. 2005). Mutation of serine 138 to arginine (S138R) in TMD III of the 5-HT2C receptor resulted in a loss of ligand binding and inositol phosphate (IP) production (Herrick-Davis et al. 2005). When HEK 293 cells were transfected with S138R, there was no detectable [3H]mesulergine binding. In addition, there was no detectable basal or 5-HT-stimulated activation of IP production, even though wild-type receptors (VSV isoform) displayed moderate levels of both basal and 5-HT-stimulated IP production. These results indicate that S138R is devoid of both ligand binding and G-protein activation. GPCR activation has been reported to involve the coordinated movements of TMDs III and VI (Farrens et al. 1996; Gether et al. 1997). The S138R mutation may directly interfere with the ability of TMD III to adopt the proper conformation to achieve an active state of the receptor. This is supported by the observation that the S138R mutation eliminates receptor basal activity.

Fluorescence confocal microscopy was used to monitor cellular trafficking patterns of wild-type and mutant S138R 5-HT2C receptors in HEK 293 cells (HerrickDavis et al. 2005). In cells expressing wild-type 5-HT2C receptors, significant constitutive receptor trafficking was observed between the plasma membrane and intracellular compartments. Within the first 3 min following the addition of 5-HT vesicular trafficking increased, followed by an overall shape change likely resulting from activation of 5-HT2C receptor activation of phospholipase D, Rho and Rac (McGrew et al. 2002). In contrast, in HEK 293 cells expressing S138R receptors there was no constitutive receptor trafficking, no receptor endocytosis in response to 5-HT, and no overall shape change during the 10-min observation period. In cells coexpressing wild-type and S138R receptors, constitutive and 5-HT-stimulated receptor trafficking was minimal. These results suggest that the ability of wild-type 5-HT2C receptors to respond to 5-HT stimulation is impaired when wild-type receptors are coexpressed with inactive S138R receptors (Herrick-Davis et al. 2005).

IP production was measured in cells coexpressing wild-type and S138R receptors to test the hypothesis that wild-type receptor function is impaired in the presence of S138R receptors. When wild-type 5-HT2C receptors were expressed in an S138R stable cell line, both basal and 5-HT-stimulated IP production were greatly reduced, with little change in 5-HT potency (Fig. 7.7a). Radioligand binding to

Fig. 7.7 (a) 5-HT dose-response curves for stimulation of 3H-IP production were measured in HEK 293 cells and the S138R stable cell line following transfection with 0.2 ug of wild-type 5-HT2C plasmid DNA. Data represent the mean ± SD of three experiments. (b) [3H]Mesulergine saturation curves in HEK 293 cells and the S138R stable cell line transfected with 0.2 or 0.5 ug of wild-type 5-HT2C plasmid DNA or vector (pcDNA3) (Reprinted from Herrick-Davis et al. 2005. With permission from ASBMB)

Fig. 7.7 (a) 5-HT dose-response curves for stimulation of 3H-IP production were measured in HEK 293 cells and the S138R stable cell line following transfection with 0.2 ug of wild-type 5-HT2C plasmid DNA. Data represent the mean ± SD of three experiments. (b) [3H]Mesulergine saturation curves in HEK 293 cells and the S138R stable cell line transfected with 0.2 or 0.5 ug of wild-type 5-HT2C plasmid DNA or vector (pcDNA3) (Reprinted from Herrick-Davis et al. 2005. With permission from ASBMB)

wild-type 5-HT2C receptors was the same following expression in HEK 293 cells and in the S138R stable cell line with no change in kilodalton or Bmax (Fig. 7.7b), indicating that the decrease IP production was not due to decreased wild-type receptor expression or binding affinity. In addition, M1-muscarinic receptors displayed normal IP signaling in the S138R cell line, demonstrating normal Gaq binding and signaling capabilities in the S138R stable cell line (Herrick-Davis et al. 2005).

Immunoprecipitation of S138R receptors following coexpression with wildtype HT2C receptors suggested that S138R may regulate the activity of wild-type receptors through a direct protein-protein interaction (Herrick-Davis et al. 2005). Therefore, live-cell acceptor photobleaching FRET experiments were performed on selected regions of plasma membrane in HEK 293 cells expressing CFP- and YFP-tagged receptors. Similar FRET efficiencies were obtained from cells expressing wild-type receptors and from cells coexpressing wild-type with S138R receptors (Herrick-Davis et al. 2005). FRET efficiency was dependent on the uD/A ratio and independent of receptor expression level. These results suggest that S138R receptors decrease the function of wild-type 5-HT2C receptors by forming inactive heterodimers that are expressed on the plasma membrane. As a result, the effective concentration of active, wild-type receptor homodimers is reduced, resulting in decreased IP signaling.

Taken together the results of these experiments indicate that the formation of a 5-HT2C heterodimer in which one protomer of the dimer is incapable of G-protein activation results in the silencing of G-protein signaling by the heterodimer. These results are consistent with a model of 5-HT2C receptor-mediated G-protein activation that requires the formation of a receptor dimer followed by the binding to and activation of a single G protein. The observation that ligand binding affinity and Bmax were unaltered following expression of wild-type receptors in the S138R cell line indicates that the formation of the wild-type/S138R heterodimer did not prevent the binding of ligand to the wild-type protomer of the heterodimer, clearly indicating that the heterodimer contains two separate ligand binding pockets. Thus the ligand/dimer/G-protein stoichiometry appears to be 2:1:1, consistent with a model in which one receptor dimer binds two molecules of ligand and one G protein (Herrick-Davis et al. 2005).

Dimer/oligomer formation may be a prerequisite for normal receptor trafficking and expression on the plasma membrane, as it may be necessary for passing ER quality control checkpoints that determine functionality. It is also possible that dimerization in the ER may be a prerequisite for trafficking to the plasma membrane as dimers may represent the basic metabotropic signaling unit. Studies involving the rhodopsin receptor support the hypothesis that the dimer may represent the basic signaling unit. Atomic force microscopy has been used to visualize rhodopsin receptors in native mammalian membranes as rows of dimeric complexes (Liang et al. 2003). In addition, the distance between the a- and g-subunits of a single heterotrimeric G protein, which are the reported regions of contact with GPCR, is predicted to be too large to accommodate a single rhodopsin receptor (Hamm 2001). Studies using chemical cross-linking and purified leukotriene B4 receptors (LTB4) have demonstrated that an LTB4 homodimer forms a pentameric complex with a single heterotrimeric G protein (Baneres and Parello 2003). In addition, the D1-D2 dopamine receptor heterodimer has been reported to form a novel signaling complex in which ligand binding to both protomers results in Gaq activation, but blockade of either protomer alone is sufficient to block signaling (O'Dowd et al. 2005). The results of these experiments are consistent with a model in which class A GPCR dimers interact with a single G protein and suggest that the dimer may represent the basic signaling unit.

Defeat Drugs and Live Free

Defeat Drugs and Live Free

Being addicted to drugs is a complicated matter condition that's been specified as a disorder that evidences in the obsessional thinking about and utilization of drugs. It's a matter that might continue to get worse and become disastrous and deadly if left untreated.

Get My Free Ebook


Post a comment