Multimeric Receptor Research Articles

Spatial organization of transmembrane receptor signalling

The spatial organization of transmembrane receptors is a critical step in signal transduction and receptor trafficking in cells. Transmembrane receptors engage in lateral homotypic and heterotypic cis-interactions as well as intercellular trans-interactions that result in the formation of signalling foci for the initiation of different signalling networks. Several aspects of ligand-induced receptor clustering and association with signalling proteins are also influenced by the lipid composition of membranes. Thus, lipid microdomains have a function in tuning the activity of many transmembrane receptors by positively or negatively affecting receptor clustering and signal transduction. We review the current knowledge about the functions of clustering of transmembrane receptors and lipid-protein interactions important for the spatial organization of signalling at the membrane.

The Signal Peptide of the IgE Receptor α-Chain Prevents Surface Expression of an Immunoreceptor Tyrosine-based Activation Motif-free Receptor Pool

The high affinity receptor for IgE, Fc epsilon receptor I (FcepsilonRI), is an activating immune receptor and key regulator of allergy. Antigen-mediated cross-linking of IgE-loaded FcepsilonRI alpha-chains induces cell activation via immunoreceptor tyrosine-based activation motifs in associated signaling subunits, such as FcepsilonRI gamma-chains. Here we show that the human FcepsilonRI alpha-chain can efficiently reach the cell surface by itself as an IgE-binding receptor in the absence of associated signaling subunits when the endogenous signal peptide is swapped for that of murine major histocompatibility complex class-I H2-K(b). This single-chain isoform of FcepsilonRI exited the endoplasmic reticulum (ER), trafficked to the Golgi and, subsequently, trafficked to the cell surface. Mutational analysis showed that the signal peptide regulates surface expression in concert with other described ER retention signals of FcepsilonRI-alpha. Once the FcepsilonRI alpha-chain reached the cell surface by itself, it formed a ligand-binding receptor that stabilized upon IgE contact. Independently of the FcepsilonRI gamma-chain, this single-chain FcepsilonRI was internalized after receptor cross-linking and trafficked into a LAMP-1-positive lysosomal compartment like multimeric FcepsilonRI. These data suggest that the single-chain isoform is capable of shuttling IgE-antigen complexes into antigen loading compartments, which plays an important physiologic role in the initiation of immune responses toward allergens. We propose that, in addition to cytosolic and transmembrane ER retention signals, the FcepsilonRI alpha-chain signal peptide contains a negative regulatory signal that prevents expression of an immunoreceptor tyrosine-based activation motif-free IgE receptor pool, which would fail to induce cell activation.

A Tyrosine Residue on the TSH Receptor Stabilizes Multimer Formation

BackgroundThe thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction.Methodology/Principal FindingsWe studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation.Conclusions/SignificanceThese data establish the TSH receptor ectodomain as one site of multimerization, independent of the transmembrane region, and that this interaction was primarily via a conserved tyrosine residue in LRR3.

119. IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA

A unique characteristic of mammalian spermatozoa is that upon ejaculation, they are unable to recognise and bind to an ovulated oocyte. These functional attributes are only realised following the sperms ascent of the female reproductive tract whereupon they undergo a myriad of biochemical and biophysical changes collectively referred to as ‘capacitation’. Since spermatozoa are both transcriptionally and translationally quiescent cells, this functional transformation must be engineered by a combination of post-translational modification and spatial reorganisation of existing sperm proteins. Indeed, evidence from our laboratory suggests that a key attribute of capacitation is the remodeling of the sperm surface architecture leading to the assembly and / or presentation of multimeric sperm-oocyte receptor complex(es). Through the novel application of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have secured the first direct evidence that human spermatozoa express a number of these protein complexes on their surface. Furthermore, we have demonstrated that a subset of these complexes harbour putative zona adhesion proteins and display strong affinity for solubilised zona pellucidae. In this study, we have extended our findings through the characterisation of one such complex containing arylsulfatase A (ASA), a protein with recognised affinity for sulfated ligands present within the zona pellucida. Through the application of immunohistochemistry and flow cytometry we revealed that ASA undergoes a capacitation-associated translocation to become expressed on the apical region of the human sperm head, a location compatible with a role in the mediation of sperm-zona pellucida interactions. This dramatic relocation was completely abolished by incubation of capacitating spermatozoa in exogenous cholesterol, suggesting that it may be driven in part by alteration in the membrane fluidity characteristics. Our current research is focused on confirming the role of ASA in human sperm-zona pellucida adhesion and elucidating the precise cellular mechanisms that underpin the proteins translocation to the cell surface.

Multivalent Supramolecular Dendrimer-Based Drugs

Supramolecular complexes consisting of a hydrophobic dendrimer host [DAB-dendr-(NHCONH-Ad)(64)] as well as solubilizing and bioactive guest molecules have been synthesized using a noncovalent approach. The guest-host supramolecular assembly is first preassembled in chloroform and transferred via the neat phase to aqueous solution. The bioactive guest molecules can bind to a natural (serotonin 5-HT(3)) receptor with nanomolar affinity as well as to the synthetic dendrimer receptor in aqueous solution, going toward a dynamic multivalent supramolecular construct capable of adapting itself to a multimeric receptor motif.

The (F)low Down on the Endothelial Epithelial Sodium Channel

The epithelial sodium channel (ENaC) contributes to blood pressure homeostasis through renal salt and water transport; increases in ENaC activity increase salt and water reabsorption along the distal nephron, increasing extracellular fluid volume and blood pressure. The importance of tubular ENaC in cardiovascular homeostasis is manifested in Liddle’s disease and type I pseudohypoaldosteronism, diseases associated with the gain and loss of ENaC function, respectively. However, several recent studies suggest that ENaC proteins may play a more ubiquitous role in blood pressure control as mechanosensors. Because ENaC proteins are closely related to a family of mechanosensitive proteins in the nematode termed “degenerins,” several laboratories have addressed the potential role of ENaC proteins as mechanosensors.1–3 In addition to their role in salt and water homeostasis, ENaC proteins and their relatives, acid-sensing ion channels (ASICs), may contribute to control of blood pressure through reflex regulation of the autonomic nervous system (baroreflex, chemoreflex, and metaboreflex) and local control of vascular tone.1 Recent literature reviews suggest that ENaC proteins may function as sensors of pressure-induced vascular stretch and laminar flow.1–3 The role of ENaC as a mediator of pressure-induced constriction in certain vessels is supported by several investigations; however, the role of ENaC as a flow sensor remains unclear.1 Because of their evolutionary relationship to mechanotransduction in the nematode, several laboratories have suggested that ENaC channels may act as flow …

The dependence on gp130 cytokines of axotomy induced neuropeptide expression in adult sympathetic neurons

Adult peripheral neurons exhibit dramatic changes in gene expression after axonal injury, including changes in neuropeptide phenotype. For example, sympathetic neurons in the superior cervical ganglion (SCG) begin to express vasoactive intestinal peptide (VIP), galanin, pituitary adenylate cyclase activating polypeptide (PACAP), and cholecystokinin after axotomy. Before these changes, nonneuronal cells in the SCG begin to express leukemia inhibitory factor (LIF). When the effects of axotomy were compared in LIF-/- and wild-type mice, the increases in VIP and galanin expression were less in the former, though significant increases still occurred. LIF belongs to a family of cytokines with overlapping physiological effects and multimeric receptors containing the subunit gp130. Real-time PCR revealed large increases in the SCG after axotomy in mRNA for three members of this cytokine family, interleukin (IL)-6, IL-11, and LIF, with modest increases in oncostatin M, no changes in ciliary neurotrophic factor, and decreases in cardiotrophin-1. To explore the role of these cytokines, animals with selective elimination of the gp130 receptor in noradrenergic neurons were studied. No significant changes in mRNA levels for VIP, galanin, and PACAP were seen in axotomized ganglia from these mutant mice, while the increase in cholecystokinin was as large as that seen in wild-type mice. The data indicate that the inductions of VIP, galanin, and PACAP after axotomy are completely dependent on gp130 cytokines and that a second cytokine, in addition to LIF, is involved. The increase in cholecystokinin after axotomy, however, does not require the action of these cytokines.

137. ELUCIDATION OF THE MOLECULAR MECHANISMS THAT UNDERPIN CAPACITATION-ASSOCIATED SPERM SURFACE REMODELLING

Recent research from our laboratory has provided evidence that sperm-egg interaction is mediated by a multimeric sperm receptor complex. Furthermore, we have demonstrated that this complex is assembled on the sperm surface during the final phase of their maturation, a process known as capacitation. The mechanisms underpinning this capacitation-associated surface remodelling remain poorly understood and are the subject of our current investigation. Specifically we have focused on whether this process is driven by vesicle mediated, intracellular trafficking of proteins. For this purpose we have examined the presence and physiological significance of a key part of the molecular machinery necessary for this form of trafficking, namely the enzyme dynamin. Our studies revealed that sperm are endowed with at least two isoforms of dynamin (1 and 2) both of which reside within the peri-acrosomal region of the sperm head, a location compatible with a role in sperm membrane remodelling. Consistent with this putative role, it was demonstrated that dynamin 1 was phosphorylated during capacitation, a post-translational modification that has been causally linked to both its activation and to the capacitation-associated surface remodelling of mouse spermatozoa. Furthermore, it was demonstrated that pharmacological inhibition of dynamin activity led to a concomitant reduction in the ability of spermatozoa to bind to the zona pellucida of homologous oocytes. This suppression was also correlated with reduced surface expression of a number of proteins including a subset of putative sperm-zona pellucida receptors. Collectively these data support the novel hypothesis that dynamin does participate indirectly in sperm membrane remodelling events by virtue of its ability to mediate intracellular trafficking.

150. THE CAPACITATION INDUCED FORMATION OF A MULTIMERIC SPERM - ZONA PELLUCIDA RECEPTOR COMPLEX

The ability of mammalian spermatozoa to fertilize the oocyte is dependent on a complex cascade of biophysical and biochemical changes collectively known as capacitation. This final phase of sperm maturation is characterised by a dramatic remodelling of the sperm surface architecture to render the cell competent to recognise and bind to the zona pellucida. Although the current paradigm suggests this interaction is mediated by a single receptor-ligand interaction, recent evidence emerging from our laboratory suggests that this event is more complex and involves the capacitation-dependent formation of a multimeric zona pellucida receptor complex. Through the novel application of Blue Native PAGE and Far Western blotting, we have recently identified the first such complex that displays the anticipated affinity for the zona pellucida. LCMS analysis revealed that this high molecular weight complex is comprised of a family of 8 chaperonin subunits that form an active t-complex polypeptide-1 (TCP-1) complex. In addition, this complex was also shown to contain an N-acetylglucosaminyltransferase, GCnT2, an enzyme with affinity for N-acetylglucosamine. Importantly, this sugar forms part of the zona pellucida glyco-matrix and has previously been implicated as a ligand for sperm interaction. Consistent with this notion, incubation of sperm with anti-GCnT2 antibodies or N-acetylglucosamine competitively inhibited sperm-zona pellucida interactions in a dose dependent manner. Collectively, this data raises the intriguing possibility that chaperonin proteins participate in the assembly and/or presentation of key zona adhesion molecules during capacitation. Future work is aimed at identifying additional zona receptors that may reside within this complex.

Extrasynaptic NR2D-Containing NMDARs Are Recruited to the Synapse during LTP of NMDAR-EPSCs

Long-term potentiation of NMDA-receptor-mediated synaptic transmission (NMDAR-LTP) is a little-understood form of plasticity. In the present study, we investigated whether NMDAR-LTP in the dentate gyrus involves recruitment of extrasynaptic NMDARs, because NMDARs are expressed both synaptically and extrasynaptically with evidence for subtype differences at different locations. We show that before induction of NMDAR-LTP, pharmacological inhibition of glutamate transporters resulted in glutamate spillover from the synapse and activation of extrasynaptic NMDARs. After the induction of NMDAR-LTP, such activation of extrasynaptic NMDARs was absent. Activation of extrasynaptic NMDARs after glutamate uptake inhibition also occurred when synaptic NMDARs were inhibited with MK801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate], and this extrasynaptically mediated NMDAR-EPSC was strongly reduced by prior induction of NMDAR-LTP. The extrasynaptic NMDARs were shown to be NR2D-containing, because the activation of extrasynaptic NMDARs by glutamate spillover was prevented by the NR2D-selective antagonists PPDA [(2R*,3S*)-1-(phenanthrenyl-2-carbonyl)piperazine-2,3-dicarboxylic acid] and UBP141. Further studies using selective antagonists for NR2A- and NR2B-containing NMDARs demonstrated that synaptic NMDARs are predominantly NR2A-containing and NR2B-containing receptors, whereas the extrasynaptic NMDARs are complex multimeric receptors with NR2A, NR2B, or NR2D subunits. Our results show that LTP of NMDAR-EPSCs involves movement of NMDARs from an extrasynaptic to a synaptic location and suggest a novel physiological role for extrasynaptic NMDARs.

In vivo supramolecular templating enhances the activity of multivalent ligands: A potential therapeutic against the Escherichia coli O157 AB 5 toxins

We demonstrate that interactions between multimeric receptors and multivalent ligands are dramatically enhanced by recruiting a complementary templating receptor such as an endogenous multimeric protein but only when individual ligands are attached to a polymer as preorganized, covalent, heterobifunctional pairs. This effect cannot be replicated by a multivalent ligand if the same recognition elements are independently arrayed on the scaffold. Application of this principle offers an approach to create high-avidity inhibitors for multimeric receptors. Judicious selection of the ligand that engages the templating protein allows appropriate effector function to be incorporated in the polymeric construct, thereby providing an opportunity for therapeutic applications. The power of this approach is exemplified by the design of exceptionally potent Escherichia coli Shiga toxin antagonists that protect transgenic mice that constitutively express a human pentraxin, serum amyloid P component.

Differential Dephosphorylation of the FcRγ Immunoreceptor Tyrosine-based Activation Motif Tyrosines with Dissimilar Potential for Activating Syk

The cell surface-expressed gamma chain of the high affinity receptor for IgE (FcepsilonRI) can be phosphorylated on two tyrosine residues of the immunoreceptor tyrosine-based activation motif (ITAM), leading to recruitment and activation of spleen tyrosine kinase (Syk), a kinase that is essential for mast cell signaling and allergic responses. However, it is not known whether preferential phosphorylation or dephosphorylation of the two individual FcRgamma tyrosines (the N-terminal Tyr47 and C-terminal Tyr58) could regulate Syk activation. Herein we report that phosphorylation of only Tyr58 was able to elicit Syk phosphorylation and a weak rise in intracellular calcium, suggesting that Tyr58 phosphorylation may be distinctively important for Syk activation. In vitro and in vivo studies revealed that both Tyr47 and Tyr58 could be similarly phosphorylated. However, mass spectrometric analysis of the phosphorylated FcepsilonRgamma from bone marrow-derived mast cells showed that phosphorylation at Tyr47 was at least 2-fold greater than at Tyr58. This suggested that, once phosphorylated, Tyr58 is preferentially dephosphorylated. In vitro studies demonstrated more efficient dephosphorylation of Tyr58 (by the receptor-associated phosphatases SHP-1 and SHP-2) than of Tyr47. Analysis of Syk binding to wild type and mutant phosphorylated FcepsilonRI revealed that mutation at Tyr58 almost completely ablated Syk binding, whereas mutation at Tyr47 moderately reduced Syk binding. The findings argue for a novel regulatory mechanism, where dephosphorylation of phospho-Tyr58 is likely to promote the down-regulation of Syk activation and suppression of mast cell responses.

Composition and significance of detergent resistant membranes in mouse spermatozoa

Mammalian spermatozoa acquire the ability to fertilize an oocyte as they ascend the female reproductive tract. This process is characterized by a complex cascade of biophysical and biochemical changes collectively know as "capacitation." The attainment of a capacitated state is accompanied by a dramatic reorganization of the surface architecture to render spermatozoa competent to recognize the oocyte and initiate fertilization. Emerging evidence indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, the mechanisms responsible for gathering key recognition molecules within this putative complex have yet to be defined. In this study, we provide the first evidence that chaperones partition into detergent resistant membrane fractions (DRMs) within capacitated mouse spermatozoa and co-localize in membrane microdomains enriched with the lipid raft marker, G(M1) ganglioside. During capacitation, these microdomains coalesce within the apical region of the sperm head, a location compatible with a role in sperm-zona pellucida interaction. Significantly, DRMs isolated from spermatozoa possessed the ability to selectively bind to the zona pellucida of unfertilized, but not fertilized, mouse oocytes. A comprehensive proteomic analysis of the DRM fractions identified a total of 100 proteins, a number of which have previously been implicated in sperm-oocyte interaction. Collectively, these data provide compelling evidence that mouse spermatozoa possess membrane microdomains that provide a platform for the assembly of key recognition molecules on the sperm surface and thus present an important mechanistic insight into the fundamental cell biological process of sperm-oocyte interaction.

Characterization of new multimeric erythropoietin receptor agonists

In addition to its natural ligand, the receptor for erythropoietin can be activated by small peptides known as erythropoietin mimetic peptides (EMPs). Although EMPs are less potent than the natural ligand, EMP dimers, consisting of two EMPs joined via a linker, have been shown to exhibit significantly improved activity compared to the corresponding monomers, with potency approaching that of the native hormone. In this study, we used a panel of novel EMP dimers to explore the effects of linker length and EMP attachment site on potency. The EC50 values obtained in an EPO-dependent proliferation assay indicated that, as has been shown with similar molecules, EMP dimerization can lead to increases in potency of more than 2 orders of magnitude. We found that both C-terminal and N-terminal attachment of the linker to EMP was tolerated, and that, with the exception of the shortest linker, all of the linker lengths tested provided a similar increase in potency. In follow-up work devised to explore the potential benefit of contacting additional cell surface EPO receptors, we designed a tetrameric template consisting of lysine-based dimers joined via commercial PEG linkers of various molecular weights. Evaluation of the resulting molecules indicated a clear effect of PEG linker size on activity, while the "dimer of dimer" with the shortest linker exhibited 10-fold lower potency than the corresponding dimer, the longest tetramer increased potency by fivefold. We discuss the implications of these results for the further development of EMP multimers.

226. Characterisation of a putative mouse sperm - zona pellucida receptor complex

Mammalian spermatozoa acquire the ability to fertilise an oocyte as they ascend the female reproductive tract. This process is characterised by a complex cascade of biophysical and biochemical changes collectively known as capacitation. The attainment of a capacitated state is accompanied by a dramatic reorganisation of the surface architecture which renders the spermatozoa competent to recognise the heterogenetic matrix of the zona pellucida surrounding the oocyte and initiate fertilisation. Emerging evidence from our laboratory indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, to date the presence and composition of such a complex has yet to be described. Through the novel application of blue native polyacrylamide gel electrophoresis (BN-PAGE), we have provided the first evidence that capacitated mouse spermatozoa express high molecular weight, multimeric protein complexes on their surface. Interestingly, at least two of these complexes contain heat shock protein 1 (HSPD1), a molecular chaperone that has previously been implicated in sperm-zona pellucida interaction. Furthermore, we were able to demonstrate that one of these complexes also possessed an affinity for solubilised zona pellucida as determined by Far-western blotting. 2D BN-PAGE was employed to further delineate the individual constituents of this high molecular weight complex, with several other chaperonin proteins not previously reported in functional sperm indentified. Collectively, these results support the notion the sperm-zona pellucida interaction are mediated by a multimeric receptor complex. Our current work is focussed on the identification of the key zona adhesion molecules that comprise this complex.

Sugar Receptors in Drosophila

The detection and discrimination of chemical compounds in potential foods are essential sensory processes when animals feed. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of mostly nonvolatile chemicals that include sugars, a diverse group of toxic compounds present in many inedible plants and spoiled foods, and pheromones [1-6]. With the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor [7-9], the functions of GRs involved in feeding are unknown. Here, we show that the Gr64 genes encode receptors for numerous sugars. We generated a fly strain that contained a deletion for all six Gr64 genes (DeltaGr64) and showed that these flies exhibit no or a significantly diminished proboscis extension reflex (PER) response when stimulated with glucose, maltose, sucrose, and several other sugars. The only considerable response was detected when Gr64 mutant flies were stimulated with fructose. Interestingly, response to trehalose is also abolished in these flies, even though they contain a functional Gr5a gene, which has been previously shown to encode a receptor for this sugar [8, 9]. This observation indicates that two or more Gr genes are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes. Finally, we present evidence that some members of the Gr64 gene family are transcribed as a polycistronic mRNA, providing a mechanism for the coexpression of multiple sugar receptors in the same taste neurons.

Multimer formation by FKBP‐12: roles for cysteine 23 and phenylalanine 36

FKBP-12 mediates the immunosuppressive actions of FK506 and rapamycin, and modulates the activities of the ryanodine, IP3 and type 1 TGF-ss receptors. Additionally, FKBP-12 possesses cis-trans peptidylprolyl isomerase (rotamase) activity. We have discovered that recombinant FKBP-12 readily forms a dimer and a small amount of trimer under nonreducing conditions. A mutant with substitution at the sole cysteine residue of FKBP-12 (C23S) did not form dimers or trimers. Using mutants with 5% or less rotamase activity, the formation of dimers was independent of enzymatic activity. The formation of trimers was abrogated by a F36Y substitution, even though dimer formation was preserved. Dimers were also observed with native FKBP-12 that was detached from rabbit skeletal muscle ryanodine receptors using FK590. The multimers of FKBP-12 could interact with molecular targets distinctly from the FKBP-12 monomer, for example, by facilitating the assembly of multimeric receptors or coordinating the activity of receptor subunits.

New developments in FcεRI regulation, function and inhibition

The high-affinity Fc receptor for IgE (FcepsilonRI), a multimeric immune receptor, is a crucial structure for IgE-mediated allergic reactions. In recent years, advances have been made in several important areas of the study of FcepsilonRI. The first area relates to FcepsilonRI-mediated biological responses that are antigen independent. The second area encompasses the biological relevance of the distinct signalling pathways that are activated by FcepsilonRI; and the third area relates to the accumulated evidence for the tight control of FcepsilonRI signalling through a broad array of inhibitory mechanisms, which are being developed into promising therapeutic approaches.

Lipid Rafts Are Triage Centers for Multimeric and Monomeric Thyrotropin Receptor Regulation

The TSH receptor (TSHR), a heptahelical G protein-coupled receptor on the surface of thyrocytes, is a major autoantigen and physiological regulator of the thyroid gland. Unlike other G protein-coupled receptors, the TSHR undergoes posttranslational cleavage of its ectodomain, leading to the existence of several forms of the receptor on the plasma membrane. We previously hypothesized that to achieve high fidelity and specificity of TSH ligand or TSHR autoantibody signaling, the TSHR may compartmentalize into microdomains within the plasma membrane. In support of this hypothesis we have shown previously that TSHRs reside in GM1 ganglioside-enriched lipid rafts in the plasma membrane of TSHR-expressing cells. In this study, we further explored the different forms of TSHRs that reside in lipid rafts. We studied both TSHR-transfected cells and rat thyrocytes, using both nondetergent biochemical analyses and receptor-lipid raft colocalization. Using the biochemical approach, we observed that monomeric receptors existed in both raft and nonraft fractions of the cell surface in the steady state. We also demonstrated that the multimeric forms of the receptor were preferentially partitioned into the lipid microdomains. Different TSHR forms, including multimers, were dynamically regulated both by receptor-specific and postreceptor-specific modulators. TSH ligand and TSHR antibody of the stimulating variety induced a decrease of multimeric forms in the raft fractions. In addition, multimeric and monomeric forms of the receptor were both associated with Gsalpha within and without the rafts. Although failure to achieve total lipid raft disruption prevented a conclusion regarding the relative power of TSHR signaling within and without the raft domains, these data showed clearly that not only were a significant proportion of TSHRs residing within lipid microdomains but that constitutive multimerization of TSHRs was actually regulated within the lipid rafts.

International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the Recognition and Nomenclature of G Protein-Coupled Receptor Heteromultimers

G protein-coupled receptors (GPCRs) have long been considered to be monomeric membrane proteins. Although numerous recent studies have indicated that GPCRs can form multimeric complexes, the functional and pharmacological consequences of this phenomenon have remained elusive. With the discovery that the functional GABA(B) receptor is an obligate heterodimer and with the use of energy transfer technologies, it is now accepted that GPCRs can form heteromultimers. In some cases, specific properties of such heteromers not shared by their respective homomers have been reported. Although in most cases these properties have only been observed in heterologous expression systems, there are a few reports describing data consistent with such heteromultimeric GPCR complexes also existing in native tissues. The present article illustrates well-documented examples of such native multimeric complexes, lists a number of recommendations for recognition and acceptance of such multimeric receptors, and gives recommendations for their nomenclature.