Controlled Dimerization of ErbB Receptors Provides Evidence for Differential Signaling by Homo- and Heterodimers

The four members of the ErbB family of receptor tyrosine kinases are involved in a complex array of combinatorial interactions involving homo- and heterodimers. Since most cell types express more than one member of the ErbB family, it is difficult to distinguish the biological activities of different homo- and heterodimers. Here we describe a method for inducing homo- or heterodimerization of ErbB receptors by using synthetic ligands without interference from the endogenous receptors. ErbB receptor chimeras containing synthetic ligand binding domains (FK506-binding protein [FKBP] or FKBP-rapamycin-binding domain [FRB]) were homodimerized with the bivalent FKBP ligand AP1510 and heterodimerized with the bifunctional FKBP-FRB ligand rapamycin. AP1510 treatment induced tyrosine phosphorylation of ErbB1 and ErbB2 homodimers and recruitment of Src homology 2 domain-containing proteins (Shc and Grb2). In addition, ErbB1 and ErbB2 homodimers activated downstream signaling pathways leading to Erk2 and Akt phosphorylation. However, only ErbB1 homodimers were internalized upon AP1510 stimulation, and only ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to form foci; however, cells expressing ErbB2 homodimers displayed a five- to sevenfold higher focus-forming ability. Using rapamycin-inducible heterodimerization we show that c-Cbl is unable to associate with ErbB1 in a ErbB1-ErbB2 heterodimer most likely because ErbB2 is unable to phosphorylate the c-Cbl binding site on ErbB1. Thus, we demonstrate that ErbB1 and ErbB2 homodimers differ in their abilities to transform fibroblasts and provide evidence for differential signaling by ErbB homodimers and heterodimers. These observations also validate the use of synthetic ligands to study the signaling and biological specificity of selected ErbB dimers in any cell type.     

Genetic analysis of the molecular basis for beta-adrenergic receptor subtype specificity

Pharmacological analysis of ligand binding to the beta-adrenergic receptor (beta AR) has revealed the existence of two distinct receptor subtypes (beta 1 and beta 2) which are the products of different genes. The predicted amino acid sequences of the beta 1 and beta 2 receptors differ by 48%. To identify the regions of the proteins responsible for determining receptor subtype, chimeras were constructed from domains of the human beta 1 and hamster beta 2 receptors. Analysis of the ligand-binding characteristics of these hybrid receptors revealed that residues in the middle portion of the beta AR sequence, particularly around transmembrane regions 4 and 5, contribute to the subtype specific binding of agonists. Smaller molecular replacements of regions of the hamster beta 2 AR with the analogous regions from the avian beta 1 AR, however, failed to identify any single residue substitution capable of altering the subtype specificity of the receptor. These data indicate that, whereas sequences around transmembrane regions 4 and 5 may contribute to conformations which influence the ligand-binding properties of the receptor, the subtype-specific differences in amine-substituted agonist binding cannot be attributed to a single molecular interaction between the ligand and any amino acid residue which is divergent between the beta 1 and beta 2 receptors.     

G protein-coupled receptor dimers: look like their parents,but act like teenagers!

Abstract

G protein-coupled receptors (GPCRs) represent the largest group of cell surface receptors and an important pharmacological target. Though originally thought to act in a one receptor–one effector fashion, it is now known that these receptors are capable of oligomerization and can function as dimers or higher order oligomers in native tissue. They do not only assemble with identical receptors as homodimers, but also associate with different GPCRs to form heterodimers. We discuss here how heterodimeric GPCRs can assemble, traffic and signal in a manner distinct from their individual receptor components or from homodimers. These receptor pairs are also demonstrated to be regulated by different chaperones, Rabs and scaffolding proteins, further emphasizing their potential as unique targets. We believe in the importance of investigating each GPCR heterodimer as an individual signaling complex, as they appear to act differently from each monomer constituting them. Just as teenagers may resemble their parents and share their genetic makeup, they can still act in a manner that is entirely unique!     

The expanding repertoire of receptor activity modifying protein (RAMP) function

Receptor activity modifying proteins (RAMPs) associate with G-protein-coupled receptors (GPCRs) at the plasma membrane and together bind a variety of peptide ligands, serving as a communication interface between the extracellular and intracellular environments. The collection of RAMP-interacting GPCRs continues to expand and now consists of GPCRs from families A, B and C, suggesting that RAMP activity is extremely prevalent. RAMP association with GPCRs can regulate GPCR function by altering ligand binding, receptor trafficking and desensitization, and downstream signaling pathways. Here, we elaborate on these RAMP-dependent mechanisms of GPCR regulation, which provide opportunities for pharmacological intervention.     

Heterodimerization of alpha 2A- and beta 1-adrenergic receptors

beta- and alpha(2)-adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the beta(1)-adrenergic receptor (beta(1)AR) and alpha(2A)-adrenergic receptor (alpha(2A)AR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta(1)AR and alpha(2A)AR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha(2A)AR was found to induce substantial internalization of coexpressed beta(1)AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [(3)H]dihydroalprenolol binding to the beta(1)AR by various ligands revealed that beta(1)AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha(2A)AR. Finally, beta(1)AR/alpha(2A)AR heterodimerization was found to be markedly enhanced by a beta(1)AR point mutation (N15A) that blocks N-linked glycosylation of the beta(1)AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha(2A)AR. These data reveal an interaction between beta(1)AR and alpha(2A)AR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.     

Homodimerization of adenosine A2A receptors: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

The results presented in this paper show that adenosine A2A receptor (A2AR) form homodimers and that homodimers but not monomers are the functional species at the cell surface. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques have been used to demonstrate in transfected HEK293 cells homodimerization of A2AR, which are heptaspanning membrane receptors with enriched expression in striatum. The existence of homodimers at the cell surface was demonstrated by time-resolved FRET. Although agonist activation of the receptor leads to the formation of receptor clusters, it did not affect the degree of A2AR-A2AR dimerization. Both monomers and dimers were detected by immunoblotting in cell extracts. However, cell surface biotinylation of proteins has made evident that more than 90% of the cell surface receptor is in its dimeric form. Thus, it seems that homodimers are the functional form of the receptor present on the plasma membrane. A deletion mutant version of the A2A receptor, lacking its C-terminal domain, was also able to form both monomeric and dimeric species when cell extracts from transfected cells were analyzed by immunoblotting. This suggests that the C-terminal tail does not participate in the dimerization. This is relevant as the C-terminal tail of A2AR is involved in heteromers formed by A2AR and dopamine D2 receptors. BRET ratios corresponding to A2AR-A2AR homodimers were higher than those encountered for heterodimers formed by A2AR and dopamine D2 receptors. As A2AR and dopamine D2 receptors do indeed interact, these results indicate that A2AR homodimers are the functional species at the cell surface and that they coexist with A2AR/D2 receptor heterodimers.     

Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis

G protein-coupled receptor (GPCR) oligomerization is a growing concept that has emerged from several studies suggesting that GPCRs can form both homo- and heterodimers. Using both coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) approaches, we established that the vasopressin V1a, V2, and the oxytocin receptors exist as homo- and hetero-dimers in transfected human embryonic kidney 293T cells. Each receptor protomer had a similar propensity to form homo- and heterodimers, indicating that their relative expression levels may determine the homo-/heterodimer ratio. The finding that immature forms of the receptor can be immunoprecipitated as homo- and heterodimers and the detection by BRET of such oligomer in endoplasmic reticulum-enriched fractions suggest that the oligomerization processes take place early during biosynthesis. Treatment with agonists or antagonists did not modify the BRET among any of the vasopressin and oxytocin receptor pairs studied, indicating that the dimerization state of the receptors is not regulated by ligand binding once they have reached the cell surface. Taken together, these results strongly support the notion that GPCR dimerization is a constitutive process.     

Homodimerization and heterodimerization of S1P/EDG sphingosine-1-phosphate receptors

Sphingosine-1-phosphate (S1P) binds to and signals through several members of a group of G protein-coupled receptors (GPCRs) known as the S1P/EDG family. Several of these receptors are coexpressed in various cell types and recent reports have shown that biological effects of S1P often require more than one S1P receptor subtype. Recent evidence indicates that many GPCRs exist as dimers. We show that S1P receptors form both homodimers as well as heterodimers with other members of the S1P subfamily of receptors. We also discuss the role that GPCR dimers play in receptor function and what this may mean for S1P signaling.     

Selective formation of ErbB-2/ErbB-3 heterodimers depends on the ErbB-3 affinity of epidermal growth factor-like ligands

EGF-like growth factors activate their ErbB receptors by promoting receptor-mediated homodimerization or, alternatively, by the formation of heterodimers with the orphan ErbB-2 through an as yet unknown mechanism. To investigate the selectivity in dimer formation by ligands, we have applied the phage display approach to obtain ligands with modified C-terminal residues that discriminate between ErbB-2 and ErbB-3 as dimerization partners. We used the epidermal growth factor/transforming growth factor alpha chimera T1E as the template molecule because it binds to ErbB-3 homodimers with low affinity and to ErbB-2/ErbB-3 heterodimers with high affinity. Many phage variants were selected with enhanced binding affinity for ErbB-3 homodimers, indicating that C-terminal residues contribute to the interaction with ErbB-3. These variants were also potent ligands for ErbB-2/ErbB-3 heterodimers despite negative selection for such heterodimers. In contrast, phage variants positively selected for binding to ErbB-2/ErbB-3 heterodimers but negatively selected for binding to ErbB-3 homodimers can be considered as "second best" ErbB-3 binders, which require ErbB-2 heterodimerization for stable complex formation. Our findings imply that epidermal growth factor-like ligands bind ErbB-3 through a multi-domain interaction involving at least both linear endings of the ligand. Apparently the ErbB-3 affinity of a ligand determines whether it can form only ErbB-2/ErbB-3 complexes or also ErbB-3 homodimers. Because no separate binding domain for ErbB-2 could be identified, our data support a model in which ErbB heterodimerization occurs through a receptor-mediated mechanism and not through bivalent ligands.     

Expression of three human beta-adrenergic-receptor subtypes in transfected Chinese hamster ovary cells.

The genes coding for three pharmacologically distinct subtypes of human beta-adrenergic receptors (beta 1 AR, beta 2 AR and beta 3 AR) were transfected for expression in Chinese hamster ovary (CHO) cells. Stable cell lines expressing each receptor were analyzed by ligand binding, adenylate cyclase activation and photoaffinity labeling, and compared to beta AR subtypes observed in previously described tissues, primary cultures and transfected cell lines. Each of the three receptor subtypes displayed saturable [125I]iodocyanopindolol-binding activity. They showed the characteristic rank order of potencies for five agonists, determined by measuring the accumulation of intracellular cAMP. These recombinant cell lines express a homogeneous population of receptors and display the known pharmacological properties of beta 1 AR and beta 2 AR, in human tissues. It is therefore likely that the pattern of ligand binding and adenylate cyclase activation, mediated by the new beta 3 AR in CHO cells, also reflects the yet-undetermined pharmacological profile in humans.     

Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling

G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays.     

Identification of an allosteric binding site for Zn2+ on the beta2 adrenergic receptor

The activity of G protein-coupled receptors (GPCRs) can be modulated by a diverse spectrum of drugs ranging from full agonists to partial agonists, antagonists, and inverse agonists. The vast majority of these ligands compete with native ligands for binding to orthosteric binding sites. Allosteric ligands have also been described for a number of GPCRs. However, little is known about the mechanism by which these ligands modulate the affinity of receptors for orthosteric ligands. We have previously reported that Zn(II) acts as a positive allosteric modulator of the beta(2)-adrenergic receptor (beta(2)AR). To identify the Zn(2+) binding site responsible for the enhancement of agonist affinity in the beta(2)AR, we mutated histidines located in hydrophilic sequences bridging the seven transmembrane domains. Mutation of His-269 abolished the effect of Zn(2+) on agonist affinity. Mutations of other histidines had no effect on agonist affinity. Further mutagenesis of residues adjacent to His-269 demonstrated that Cys-265 and Glu-225 are also required to achieve the full allosteric effect of Zn(2+) on agonist binding. Our results suggest that bridging of the cytoplasmic extensions of TM5 and TM6 by Zn(2+) facilitates agonist binding. These results are in agreement with recent biophysical studies demonstrating that agonist binding leads to movement of TM6 relative to TM5.     

Constitutive homo- and hetero-oligomerization of TbetaRII-B, an alternatively spliced variant of the mouse TGF-beta type II receptor

Transforming growth factor (TGF)-beta ligands signal through transmembrane type I and type II serine/threonine kinase receptors, which form heteromeric signalling complexes upon ligand binding. Type II TGF-beta receptors (TbetaRII) are reported to exist as homodimers at the cell surface, but the oligomerization pattern and dynamics of TbetaRII splice variants in live cells has not been demonstrated thus far. Using co-immunoprecipitation and bioluminescence resonance energy transfer (BRET), we demonstrate that the mouse TbetaRII receptor splice variant TbetaRII-B is capable of forming ligand-independent homodimers and heterodimers with TbetaRII. The homomeric interaction of mouse (m)TbetaRII-B isoforms, however, is less robust than the heteromeric interactions of mTbetaRII-B with wild-type TbetaRII, which indicates that these receptors may be more likely to heterodimerize when both receptors are expressed. Moreover, we demonstrate that mTbetaRII-B is a signalling receptor with ubiquitous tissue expression. Our study thus demonstrates previously unappreciated complex formation of TGF-beta type II receptors, and suggests that mTbetaRII-B can direct TGF-beta-induced signalling in vitro and in vivo.     

Selective binding of ligands to beta 1, beta 2 or chimeric beta 1/beta 2-adrenergic receptors involves multiple subsites.

The molecular basis of ligand binding selectivity to beta-adrenergic receptor subtypes was investigated by designing chimeric beta 1/beta 2-adrenergic receptors. These molecules consisted of a set of reciprocal constructions, obtained by the exchange between the wild-type receptor genes of one to three unmodified transmembrane regions, together with their extracellular flanking regions. Eight different chimeras were expressed in Escherichia coli and studied with selective beta-adrenergic ligands. The evaluation of the relative effect of each chimeric exchange on ligand binding affinity was based on the analysis of delta G values, calculated from the equilibrium binding constants, as a function of the number of substituted beta 2-adrenergic receptor transmembrane domains. The data showed that the contribution of each exchanged region to subtype selectivity varies with each ligand; moreover, while several regions are critical for the pharmacological selectivity of all ligands, others are involved in the selectivity of only some compounds. The selectivity displayed by beta-adrenergic compounds towards beta 1 or beta 2 receptor subtypes thus results from a particular combination of interactions between each ligand and each of the subsites, variably distributed over the seven transmembrane regions of the receptor; these subsites are presumably defined by the individual structural properties of the ligands.     

Ligand-Specific Interactions Modulate Kinetic, Energetic, and Mechanical Properties of the Human β(2) Adrenergic Receptor

G protein-coupled receptors (GPCRs) are a class of versatile proteins that transduce signals across membranes. Extracellular stimuli induce inter- and intramolecular interactions that change the functional state of GPCRs and activate intracellular messenger molecules. How these interactions are established and how they modulate the functional state of GPCRs remain to be understood. We used dynamic single-molecule force spectroscopy to investigate how ligand binding modulates the energy landscape of the human β(2) adrenergic receptor (β(2)AR). Five different ligands representing either agonists, inverse agonists or neutral antagonists established a complex network of interactions that tuned the kinetic, energetic, and mechanical properties of functionally important structural regions of β(2)AR. These interactions were specific to the efficacy profile of the ligands investigated and suggest that the functional modulation of GPCRs follows structurally well-defined interaction patterns.     

Melanocortin receptors form constitutive homo- and heterodimers

A significant number of G protein-coupled receptors are shown to form homo- or heterodimers/oligomers, and oligomerization of GPCRs may be a quite general phenomenon. We have here explored the possibility that the two closely related human melanocortin receptor 1 (MC(1)R) and melanocortin receptor 3 (MC(3)R) form dimers. Using bioluminescence resonance energy transfer (BRET(2)) we demonstrate that MC(1) and MC(3)Rs form homo- and heterodimers, when expressed in Cos-7 cells. Treatment with agonist, partial agonist or antagonists did not modify the BRET(2) signal for any of the receptor pairs studied, suggesting that the dimerization is not regulated by ligand binding. Rather our results indicate that melanocortin receptors exist as constitutively pre-formed dimers.     

NHERF-1 and the cytoskeleton regulate the traffic and membrane dynamics of G protein-coupled receptors

The sodium-hydrogen exchange regulatory factor 1 (NHERF-1/EBP50) interacts with the C terminus of several G protein-coupled receptors (GPCRs). We examined the role of NHERF-1 and the cytoskeleton on the distribution, dynamics, and trafficking of the beta(2)-adrenergic receptor (beta(2)AR; a type A receptor), the parathyroid hormone receptor (PTH1R; type B), and the calcium-sensing receptor (CaSR; type C) using fluorescence recovery after photobleaching, total internal reflection fluorescence, and image correlation spectroscopy. beta(2)AR bundles were observed only in cells that expressed NHERF-1, whereas the PTH1R was localized to bundles that parallel stress fibers independently of NHERF-1. The CaSR was never observed in bundles. NHERF-1 reduced the diffusion of the beta(2)AR and the PTH1R. The addition of ligand increased the diffusion coefficient and the mobile fraction of the PTH1R. Isoproterenol decreased the immobile fraction but did not affect the diffusion coefficient of the beta(2)AR. The diffusion of the CaSR was unaffected by NHERF-1 or the addition of calcium. NHERF-1 reduced the rate of ligand-induced internalization of the PTH1R. This phenomenon was accompanied by a reduction of the rate of arrestin binding to PTH1R in ligand-exposed cells. We conclude that some GPCRs, such as the beta(2)AR, are attached to the cytoskeleton primarily via the binding of NHERF-1. Others, such as the PTH1R, bind the cytoskeleton via several interacting proteins, one of which is NHERF-1. Finally, receptors such as the CaSR do not interact with the cytoskeleton in any significant manner. These interactions, or the lack thereof, govern the dynamics and trafficking of the receptor.     

Epidermal growth factor mutant with wild-type affinity for both ErbB1 and ErbB3

The family of epidermal growth factor (EGF)-like ligands binds to ErbB receptors in a highly selective manner. Previous studies indicated that both linear regions of the ligand play a major role in determining receptor selectivity, and phage display studies showed that each region could be optimized independently for enhanced affinity. In this study, we broadened the ErbB binding specificity of EGF by introducing the optimal sequence requirements for ErbB3 binding in both the N- and C-terminal linear regions. One such EGF mutant, designated WVR/EGF/IADIQ, gained high affinity for ErbB3 and showed concomitant ErbB3 activation through ErbB2.ErbB3 heterodimers similar to the natural ErbB3 ligand NRG1beta, while the capacity to bind and activate ErbB1 was fully maintained. Despite its high affinity for ErbB1 and ErbB3, this mutant was unable to activate ErbB1.ErbB3 heterodimers, as shown by the cell survival and receptor phosphorylation analysis. We concluded that despite the fact that no naturally occurring ligand exists with this dual-specificity, high-affinity binding to both ErbB1 and ErbB3 is not mutually exclusive. This mutant can be useful in a direct structural comparison of the ligand-binding characteristics of ErbB1 and ErbB3.