The Conserved Arginine Cluster in the Insert of the Third Cytoplasmic Loop of the Long Form of the D2 Dopamine Receptor (D2L-R) Acts as an Intracellular Retention Signal.

This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D2 dopamine receptor (D2L-R) confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR) located within the insert could act as an RXR-type endoplasmic reticulum (ER) retention signal. Arginine residues (R) within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E), either individually or in clusters, thus generating single, double, and triple D2L-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA), radioligand binding assay, bioluminescence resonance energy transfer (BRET²) ß-arrestin 2 (ßarr2) recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E) substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D2L-R mutants preserved their functional integrity regarding ligand binding, agonist-induced ßarr2 recruitment and Gαi-mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3) of the D2L-R appears to be the ER retention signal.

Molecular determinants of orexin receptor-arrestin-ubiquitin complex formation.

BACKGROUND AND PURPOSE: The orexin system regulates a multitude of key physiological processes, particularly involving maintenance of metabolic homeostasis. Consequently, there is considerable potential for pharmaceutical development for the treatment of disorders from narcolepsy to metabolic syndrome. It acts through the hormonal activity of two endogenous peptides, orexin A binding to orexin receptors 1 and 2 (OX1 and OX2) with similar affinity, and orexin B binding to OX2 with higher affinity than OX1 receptors. We have previously revealed data differentiating orexin receptor subtypes with respect to their relative stability in forming orexin receptor-arrestin-ubiquitin complexes measured by BRET. Recycling and cellular signalling distinctions were also observed. Here, we have investigated, using BRET, the molecular determinants involved in providing OX2 receptors with greater ß-arrestin-ubiquitin complex stability. EXPERIMENTAL APPROACH: The contribution of the C-terminal tail of the OX receptors was investigated by bulk substitution and site-specific mutagenesis using BRET and inositol phosphate assays. KEY RESULTS: Replacement of the OX1 receptor C-terminus with that of the OX2 receptor did not result in the expected gain of function, indicating a role for intracellular domain configuration in addition to primary structure. Furthermore, two out of the three putative serine/threonine clusters in the C-terminus were found to be involved in OX2 receptor-ß-arrestin-ubiquitin complex formation. CONCLUSIONS AND IMPLICATIONS: This study provides fundamental insights into the molecular elements that influence receptor-arrestin-ubiquitin complex formation. Understanding how and why the orexin receptors can be functionally differentiated brings us closer to exploiting these receptors as drug targets.

Temporal profiling of orexin receptor-arrestin-ubiquitin complexes reveals differences between receptor subtypes.

Orexin G protein-coupled receptors (OxRs) and their cognate agonists have been implicated in a number of disorders since their recent discovery, ranging from narcolepsy to formation of addictive behavior. Bioluminescence resonance energy transfer assays of agonist-occupied OxRs provided evidence for a strong dose-dependent interaction with both trafficking proteins ß-arrestin 1 and 2 that required unusually high agonist concentrations compared with inositol phosphate signaling. This appears to be reflected in functional differences in potency with respect to orexin A (OxA) and OxR2-dependent ERK1/2 phosphorylation after 90 min compared with 2 min, potentially consistent with ß-arrestin-mediated versus G protein-mediated signaling, respectively. Furthermore, extended bioluminescence resonance energy transfer kinetic data monitoring OxA-dependent receptor-ß-arrestin and ß-arrestin-ubiquitin proximity suggested subtype-specific differences in receptor trafficking, with OxR2 activation resulting in more sustained receptor-ß-arrestin-ubiquitin complex formation than elicited by OxR1 activation. Enzyme-linked immunosorbent assay (ELISA) data also revealed that OxR1 underwent significantly more rapid recycling compared with OxR2. Finally, we have observed sustained OxA-dependent ERK1/2 phosphorylation in the presence of OxR2 compared with OxR1. Although both OxR subtypes could be classified as class B receptors for ß-arrestin usage based on the initial strength of interaction with both ß-arrestins, our temporal profiling revealed tangible differences between OxR subtypes. Consequently, OxR1 appears to fit uneasily into the commonly used ß-arrestin classification scheme. More importantly, it is hoped that this improved profiling capability, enabling the subtleties of protein complex formation, stability, and duration to be assessed in live cells, will help unlock the therapeutic potential of targeting these receptors.

Application of G protein-coupled receptor-heteromer identification technology to monitor ß-arrestin recruitment to G protein-coupled receptor heteromers.

Understanding the role of G protein-coupled receptor (GPCR; also known as a 7 transmembrane receptor) heteromerization in the physiology and pathophysiology of cellular function has now become a major research focus. However, there is currently a lack of cell-based assays capable of profiling the specific functional consequences of heteromerization in a ligand-dependent manner. Understanding the pharmacology specifically associated with heteromer function in contrast to monomer or homomer function enables the so-called biochemical fingerprints of the receptor heteromer to be ascertained. This is the first step in establishing the physiological relevance of heteromerization, the goal of everyone in the field, as these fingerprints can then be utilized in future endeavors to elucidate heteromer function in native tissues. The simple, robust, ligand-dependent methodology described in this study utilizes a novel configuration of components of a proximity-based reporter system. This is exemplified by the use of bioluminescence resonance energy transfer due to the advantages of real-time live cell monitoring of proximity specifically between the heteromer complex and a protein that is recruited in a ligand-dependent manner, in this case, ß-arrestin 2. Further, the demonstration of Z'-factor values in excess of 0.6 shows the potential of the method for screening compounds for heteromer-selective or biased activity. Three previously characterized GPCR heteromers, the chemokine receptor heteromers CCR2-CCR5 and CCR2-CXCR4, as well as the angiotensin II receptor type 1-bradykinin receptor type 2 heteromer, have been used to illustrate the profiling capability and specificity of the GPCR heteromer identification technology.

Agonist-independent interactions between beta-arrestins and mutant vasopressin type II receptors associated with nephrogenic syndrome of inappropriate antidiuresis.

Nephrogenic syndrome of inappropriate antidiuresis is a recently identified genetic disease first described in two unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. It was found that each infant had a different mutation of the vasopressin type II receptor (V2R) at codon 137 where arginine was converted to cysteine or leucine (R137C or R137L), resulting in constitutive signaling. Interestingly, a missense mutation at the same codon, converting arginine to histidine (R137H), leads to the opposite disease phenotype with a loss of the kidney's ability to concentrate urine resulting in nephrogenic diabetes insipidus. This mutation is associated with impaired signaling, although whether this is predominantly due to impaired trafficking to the plasma membrane, agonist-independent internalization, or G protein uncoupling is currently unclear. Using bioluminescence resonance energy transfer and confocal microscopy, we demonstrate that both V2R-R137C and V2R-R137L mutants interact with beta-arrestins in an agonist-independent manner resulting in dynamin-dependent internalization. This phenotype is similar to that observed for V2R-R137H, which is intriguing considering that it is accompanied by constitutive rather than impaired signaling. Consequently, it would seem that agonist-independent internalization per se is unlikely to be the major determinant of impaired V2R-R137H signaling. Our findings indicate that the V2R-R137C and V2R-R137L mutants traffic considerably more efficiently to the plasma membrane than V2R-R137H, identifying this as a potentially important mutation-dependent difference affecting V2R function.

Demonstration of improvements to the bioluminescence resonance energy transfer (BRET) technology for the monitoring of G protein-coupled receptors in live cells.

The bioluminescence resonance energy transfer (BRET) technique has become extremely popular for studying protein-protein interactions in living cells and real time. Of particular interest is the ability to monitor interactions between G protein-coupled receptors, such as the thyrotropin-releasing hormone receptor (TRHR), and proteins critical for regulating their function, such as beta-arrestin. Using TRHR/beta-arrestin interactions, we have demonstrated improvements to all 3 generations of BRET (BRET(1), BRET(2), and eBRET) by using the novel forms of luciferase, Rluc2 and Rluc8, developed by the Gambhir laboratory. Furthermore, for the 1st time it was possible to use the BRET2 system to detect ligand-induced G protein-coupled receptor/beta-arrestin interactions over prolonged periods (on the scale of hours rather than seconds) with a very stable signal. As demonstrated by our Z'-factor data, these luciferases increase the sensitivity of BRET to such an extent that they substantially increase the potential applicability of this technology for effective drug discovery high-throughput screening.

G protein-coupled receptor dimers: functional consequences, disease states and drug targets.

With an ever-expanding need for reliable therapeutic agents that are highly effective and exhibit minimal deleterious side effects, a greater understanding of the mechanisms underlying G protein-coupled receptor (GPCR) regulation is fundamental. GPCRs comprise more than 30% of all therapeutic drug targets and it is likely that this will only increase as more orphan GPCRs are identified. The past decade has seen a dramatic shift in the prevailing concept of how GPCRs function, in particular the growing acceptance that GPCRs are capable of interacting with one another at a molecular level to form complexes, with significantly different pharmacological properties to their monomeric selves. While the ability of like-receptors to associate and form homodimers raises some interesting mechanistic issues, the possibility that unlike-receptors could heterodimerise in certain tissue types, producing a functionally unique signalling complex that binds specific ligands, provides an invaluable opportunity to refine and redefine pharmacological interventions with greater specificity and efficacy.

The duffy antigen/receptor for chemokines exists in an oligomeric form in living cells and functionally antagonizes CCR5 signaling through hetero-oligomerization.

The Duffy antigen/receptor for chemokines (DARC) is an unusual chemokine receptor that binds a large number of inflammatory chemokines of both the CC and CXC families with nanomolar affinity, yet it lacks the ability to signal upon ligand binding. Using bioluminescent resonant energy transfer, we have demonstrated for the first time that DARC exists as a constitutive homo-oligomer in living cells and furthermore that DARC hetero-oligomerizes with the CC chemokine receptor CCR5. DARC-CCR5 interaction impairs chemotaxis and calcium flux through CCR5, whereas internalization of CCR5 in response to ligand binding remains unchanged. These results suggest a novel mechanism by which DARC could modulate inflammatory responses to chemokines in vivo.

Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption.

Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H+)-ATPases (V-ATPases) polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V(1) and V(0). The peripheral cytoplasmically oriented V(1) domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V(0) domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V(0) domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c'', and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c'', and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V(0) domain and efficient osteoclastic bone resorption.

Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET).

Bioluminescence resonance energy transfer (BRET) is a straightforward biophysical technique for studying protein-protein interactions. It requires: (1) that proteins of interest and suitable controls be labeled with either a donor or acceptor molecule, (2) placement of these labeled proteins in the desired environment for assessing their potential interaction, and (3) use of suitable detection instrumentation to monitor resultant energy transfer. There are now several possible applications, combinations of donor and acceptor molecules, potential assay environments and detection system perturbations. Therefore, this review aims to demystify and clarify the important aspects of the BRET methodology that should be considered when using this technique.

Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells.

Bioluminescence resonance energy transfer (BRET) is an increasingly popular technique for studying protein-protein interactions in live cells. It is particularly suitable for real-time monitoring of such interactions, however, the timescale over which assays can be carried out is currently relatively short (minutes) due to substrate instability. We present a new derivation of the BRET technology, termed 'extended BRET' (eBRET), which now enables protein-protein interactions to be monitored in real-time for many hours. This capability has significant benefits for investigating cellular function over extended timescales, as we have illustrated using the agonist-induced G-protein coupled receptor/beta-arrestin interaction. The potential for studying the modulation of such interactions by agonists, antagonists, inhibitors, dominant negative mutants and co-expressed accessory proteins is substantial. Furthermore, the advantages of eBRET have important implications for the development of high-throughput BRET screening systems, an ever-expanding area of interest for the pharmaceutical industry.

Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions.

A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol encompasses BRET1, BRET2 and the recently described eBRET, including selection of the donor, acceptor and substrate combination, fusion construct generation and validation, cell culture, fluorescence and luminescence detection, BRET detection and data analysis. The protocol is particularly suited to studying protein-protein interactions in live cells (adherent or in suspension), but cell extracts and purified proteins can also be used. Furthermore, although the procedure is illustrated with references to mammalian cell culture conditions, this protocol can be readily used for bacterial or plant studies. Once fusion proteins are generated and validated, the procedure typically takes 48-72 h depending on cell culture requirements.

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer.

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

Helix I of beta-arrestin is involved in postendocytic trafficking but is not required for membrane translocation, receptor binding, and internalization.

beta-Arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors (GPCRs), including the type 1 angiotensin II receptor (AT(1)R), to promote receptor desensitization and internalization. The AT(1) R is a class B GPCR that recruits both beta-arrestin1 and beta-arrestin2, forming stable complexes that cotraffic to deep-core endocytic vesicles. beta-Arrestins contain one amphipathic and potentially amphitropic (membrane-targeting) alpha-helix (helix I) that may promote translocation to the membrane or influence receptor internalization or trafficking. Here, we investigated the trafficking and function of beta-arrestin1 and beta-arrestin2 mutants bearing substitutions in both the hydrophobic and positively charged faces of helix I. The level of expression of these mutants and their cytoplasmic localization (in the absence of receptor activation) was similar to wild-type beta-arrestins. After angiotensin II stimulation, both wild-type and beta-arrestin mutants translocated to the cell membrane, although recruitment was weaker for mutants of the hydrophobic face of helix I. For all beta-arrestin mutants, the formation of deep-core vesicles was less observed compared with wild-type beta-arrestins. Furthermore, helix I conjugated to green fluorescent protein is not membrane-localized, suggesting that helix I, in isolation, is not amphitropic. Bioluminescence resonance energy transfer analysis revealed that both wild-type and beta-arrestin mutants retained a capacity to interact with the AT(1)R, although the interaction with the mutants was less stable. Finally, wild-type and mutant beta-arrestins fully supported receptor internalization in human embryonic kidney cells and mouse embryonic fibroblasts deficient in beta-arrestin1 and -2. Thus, helix I is implicated in postmembrane trafficking but is not strongly amphitropic.

Expression of orexin receptors in the brain and peripheral tissues of the male sheep.

Orexins exert their effects through two specific receptors (OX1R and OX2R) that have been found mainly in the brain and also in peripheral tissues of rats and humans. Here, we demonstrate expression of mRNA encoding for ovine OX1R and OX2R in central and peripheral tissues of sheep. Gene expression for orexin receptors in the hypothalamus and the preoptic area was localised by in situ hybridisation. OX1R was detected in arcuate nuclei (ARC), median eminence (ME), the lateral hypothalamic nuclei and preoptic area (POA) and it was scattered along the third ventricle from the paraventricular (PVN) to the ventromedial hypothalamic nuclei (VMH). OX2R was localised in the PVN, ARC, ME, ventral VMH and a small region of the ventral POA. Gene expression for OX1R and OX2R in central and peripheral tissues was analysed using quantitative real time RT-PCR. Both orexin receptor genes were expressed in the hypothalamus, POA, hippocampus, amygdala, olfactory bulb, pineal gland and recess and pituitary gland, whereas only OX1R mRNA was detected in the testis, kidney and adrenal gland. The expression of the genes for orexin receptors in this range of ovine tissues suggests roles for orexins in multiple physiological functions, with actions at both central and peripheral levels.

Monitoring the formation of dynamic G-protein-coupled receptor-protein complexes in living cells.

GPCRs (G-protein-coupled receptors) play an extremely important role in transducing extracellular signals across the cell membrane with high specificity and sensitivity. They are central to many of the body's endocrine and neurotransmitter pathways, and are consequently a major drug target. It is now clear that GPCRs interact with a range of proteins, including other GPCRs. Identifying and elucidating the function of such interactions will significantly enhance our understanding of cellular function, with the promise of new and improved pharmaceuticals. Biophysical techniques involving resonance energy transfer, namely FRET (fluorescence resonance energy transfer) and BRET (bioluminescence resonance energy transfer), now enable us to monitor the formation of dynamic GPCR-protein complexes in living cells, in real time. Their use has firmly established the concept of GPCR oligomerization, as well as demonstrating GPCR interactions with GPCR kinases, beta-arrestins, adenylate cyclase and a subunit of an inwardly rectifying K+ channel. The present review examines recent technological advances and experimental applications of FRET and BRET, discussing particularly how they have been adapted to extract an ever-increasing amount of information about the nature, specificity, stoichiometry, kinetics and agonist-dependency of GPCR-protein interactions.

Functional deletion of the calcium-sensing receptor in a case of neonatal severe hyperparathyroidism.

Heterozygous inactivating mutations of the calcium-sensing receptor (CaR) cause familial hypocalciuric hypercalcemia, whereas homozygous or compound heterozygous inactivating mutations normally cause neonatal severe hyperparathyroidism. In a case of neonatal severe hyperparathyroidism characterized by moderately severe hypercalcemia and very high PTH levels, coupled with evidence of hyperparathyroidism and effects on brain development not previously demonstrated, we detected point mutations on separate alleles of the CaR, resulting in premature stop codon substitutions at G94 and R648. This led to severely truncated receptors and an effective so-called knockout of functional CaR. FLAG-tagged, truncated receptors were expressed in HEK293 cells for functional analysis. Confocal microscopy demonstrated cytoplasmic localization of the G94stop receptor, whereas the R648stop receptor was present both in the cytoplasm and associated with the cell membrane. Only the R648stop receptor could be detected by Western analysis. Functional assays in which R648stop and wild-type receptor were cotransfected into HEK293 cells demonstrated a reduction in wild-type Ca(2+)-responsiveness by the R648stop receptor, even at physiological Ca(2+) levels, thus simulating familial hypocalciuric hypercalcemia in relatives of the infant who were heterozygous for the R648stop mutation. The R648stop receptor alone was nonresponsive to Ca(2+). This case contributes to our understanding of the clinical manifestation of a CaR knockout.

Study of G-protein-coupled receptor-protein interactions by bioluminescence resonance energy transfer.

Complex networks of protein-protein interactions are key determinants of cellular function, including those regulated by G-protein-coupled receptors (GPCRs). Formation of either stable or transitory complexes are involved in regulating all aspects of receptor function, from ligand binding through to signal transduction, desensitization, resensitization and downregulation. Today, 50% of all recently launched drugs are targeted against GPCRs. This particular class of proteins is extremely useful as a drug target because the receptors are partly located outside the cell, simplifying bioavailability and delivery of drugs directed against them. However, being located within the cell membrane causes difficulties for the study of GPCR function and bioluminescence resonance energy transfer (BRET), a naturally occurring phenomenon, represents a newly emerging, powerful tool with which to investigate and monitor dynamic interactions involving this receptor class. BRET is a noninvasive, highly sensitive technique, performed as a simple homogeneous assay. involving the proximity-dependent transfer of energy from an energy donor to acceptor resulting in the emission of light. This technology has several advantages over alternative approaches as the detection occurs within live cells, in real time, and is not restricted to a particular cellular compartment. The use of such biophysical techniques as BRET, will not only increase our understanding of the nature of GPCR regulation and the protein complexes involved, but could also potentially lead to the development of novel therapeutics that modulate these interactions.

alpha(v)beta(3) Integrin interacts with the transforming growth factor beta (TGFbeta) type II receptor to potentiate the proliferative effects of TGFbeta1 in living human lung fibroblasts.

The alpha(v)beta(3) integrin is known to cooperate with receptor tyrosine kinases to enhance cellular responses. To determine whether alpha(v)beta(3) regulates transforming growth factor beta (TGFbeta) 1-induced responses, we investigated the interaction between alpha(v)beta(3) and TGFbeta type II receptor (TGFbetaIIR) in primary human lung fibroblasts. We report that TGFbeta1 up-regulates cell surface and mRNA expression of alpha(v)beta(3) in a time- and dose-dependent manner. Co-immunoprecipitation and confocal microscopy showed that TGFbetaRII associates and clusters with alpha(v)beta(3), following TGFbeta1 exposure. This association was not observed with alpha(v)beta(5) or alpha(5)beta(1). We also used a novel molecular proximity assay, bioluminescence resonance energy transfer (BRET), to quantify this dynamic interaction in living cells. TGFbeta1 stimulation resulted in a BRET signal within 5 min, whereas tenascin, which binds alpha(v)beta(3), did not induce a substantial BRET signal. Co-exposure to tenascin and TGFbeta1 produced no further increases in BRET than TGFbeta1 alone. Cyclin D1 was rapidly induced in cells co-exposed to TGFbeta1 and tenascin, and as a consequence proliferation induced by TGFbeta1 was dramatically enhanced in cells co-exposed to tenascin or vitronectin. Cholesterol depletion inhibited the interaction between TGFbetaRII and alpha(v)beta(3) and abrogated the proliferative effect. The cyclic RGD peptide, GpenGRGDSPCA, which blocks alpha(v)beta(3), also abolished the synergistic proliferative effect seen. These results indicate a new interaction partner for the alpha(v)beta(3) integrin, the TGFbetaIIR, in which TGFbeta1-induced responses are potentiated in the presence alpha(v)beta(3) ligands. Our data provide a novel mechanism by which TGFbeta1 may contribute to abnormal wound healing and tissue fibrosis.

Receptors for hypothalamic releasing hormones TRH and GnRH: oligomerization and interactions with intracellular proteins.

Studies of TRH and GnRH receptors have revealed much information about the roles of G-proteins and beta-arrestins, as well as receptor residues important for signaling, desensitization and internalization. However, the proteins involved are only just beginning to be identified and characterized. Additional complexity now exists with the observation that these receptors form oligomers in live cells. Indeed, hetero-oligomerization of TRH receptor subtypes 1 and 2 potentially alters interactions with intracellular regulatory proteins. Knowledge of proteins that interact with TRH or GnRH receptors will increase our understanding of receptor function and provide potential drug targets for a range of receptor-associated conditions.