Molecular Mechanisms of Phospholipase C β3 Autoinhibition

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Semaphorin 4D Signaling Requires the Recruitment of Phospholipase Cγ into the Plexin-B1 Receptor Complex

The semaphorin 4D (Sema4D) receptor plexin-B1 constitutively interacts with particular Rho guanine nucleotide exchange factors (RhoGEFs) and thereby mediates Sema4D-induced RhoA activation, a process which involves the tyrosine phosphorylation of plexin-B1 by ErbB-2. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGEF activity. We show here that activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation creates docking sites for the SH2 domains of phospholipase Cγ (PLCγ). PLCγ is thereby recruited into the plexin-B1 receptor complex and via its SH3 domain activates the Rho guanine nucleotide exchange factor PDZ-RhoGEF. PLCγ-dependent RhoGEF activation is independent of its lipase activity. The recruitment of PLCγ has no effect on the R-Ras GTPase-activating protein activity of plexin-B1 but is required for Sema4D-induced axonal growth cone collapse as well as for the promigratory effects of Sema4D on cancer cells. These data demonstrate a novel nonenzymatic function of PLCγ as an important mechanism of plexin-mediated signaling which links tyrosine phosphorylation of plexin-B1 to the regulation of a RhoGEF protein and downstream cellular processes.Mammalian semaphorins were originally identified as axon guidance factors but are now recognized also as important regulators of morphogenesis and homeostasis in various organ systems, including the immune, cardiovascular, and renal systems (3-5, 7, 19, 23, 30, 35, 40, 56, 64, 76). Most effects of semaphorins are mediated by a group of large transmembrane proteins called plexins, of which four families exist in the mammalian system: plexin-A1 to -4, plexin-B1 to -3, plexin-C1, and plexin-D1 (60, 61). The four members of the plexin-A family in most cases require neuropilins as ligand binding partners to respond to semaphorins, whereas the three members of the plexin-B family are directly activated by semaphorins. While plexin-B1 binds Sema4D, plexin-B2 can be activated by Sema4C and Sema4D, and plexin-B3 has been shown to respond to Sema5A (31, 35).The activation of plexins by semaphorins initiates a variety of signaling processes, which involve several small GTPases of the Ras and Rho families (31, 34, 43). All plexin family members possess an R-Ras GTPase-activating protein (GAP) domain (36). Activated plexin-B1 and -A1 have been shown to also interact with other small GTPases, including GTP-bound Rac1 and RhoD as well as Rnd1, Rnd2, and Rnd3 (14, 37, 48, 63, 67, 68, 74). Different from other plexin families, the C terminus of B-family plexins contains a PDZ domain-binding motif which mediates a stable interaction with the guanine nucleotide exchange factors PDZ-RhoGEF and LARG (1, 15, 26, 39, 57). Activation of the plexin-B1/PDZ-RhoGEF complex by semaphorin 4D (Sema4D) results in RhoA activation downstream of plexin-B1 (15, 39, 57). Members of the plexin-B family also interact with and are phosphorylated by the receptor tyrosine kinases ErbB-2 and c-Met (12, 22, 58). ErbB-2-mediated phosphorylation of plexin-B1 is required for plexin-mediated RhoA activation and downstream cellular effects, including the promigratory effects of Sema4D on cancer cells and the induction of axonal growth cone collapse by Sema4D (58, 59). However, the molecular mechanisms linking ErbB-2-mediated phosphorylation of plexin-B1 to the regulation of RhoA activity and subsequent cellular effects are unknown.Here we report that upon activation by Sema4D, plexin-B1 becomes phosphorylated by ErbB-2 at particular tyrosine residues on its intracellular portion. These phosphorylated tyrosine residues serve as docking sites for the SH2 domains of PLCγ. PLCγ is thereby recruited into the plexin-B1 receptor complex and through its SH3 domain mediates RhoA activation and downstream cellular effects.     

Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity

Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn''s activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn''s dynamics at the PM and its relation to its inhibition of exocytosis.     

Binding of Chimeric Peptides M617 and M871 to Galanin Receptor Type 3 Reveals Characteristics of Galanin Receptor–Ligand Interaction

The neuropeptide galanin is ascribed to a variety of biological effects, but selective compounds to examine the specific roles of the three receptor subtypes are currently lacking. The recently introduced chimeric peptide ligands M617 and M871 target the galanin receptors GalR1 and GalR2, respectively. These peptides have been used to examine receptor function in vitro and in vivo, but their affinity to GalR3 has not been tested. Here, we report the binding affinity of these peptides at human GalR3 and demonstrate that M617 binds GalR3 and stimulates this receptor in an agonistic manner, whereas M871 shows very low affinity towards GalR3 (K _i 49.2 ± 9.4 nM and >10 μM, respectively). An l-alanine scan of M617 revealed the importance of the ligand C-terminus in GalR3 binding, which stands in contrast to the structural requirements for binding to GalR1 and GalR2. These data provide insights into galanin receptor ligand binding that should be considered when using these compounds in functional studies.     

Use of M1–M5 Muscarinic Receptor Knockout Mice as Novel Tools to Delineate the Physiological Roles of the Muscarinic Cholinergic System

In this review we report recent findings on the physiological role of the five known muscarinic acetylcholine receptors (mAChRs) as shown by gene targeting technology. Using knockout mice for each mAChRs subtype, the role of mAChRs subtypes in a number of physiological functions was confirmed and new activities were discovered. The M₁ mAChRs modulate neurotransmitter signaling in cortex and hippocampus. The M₃ mAChRs are involved in exocrine gland secretion, smooth muscle contractility, pupil dilation, food intake, and weight gain. The role of the M₅ mAChRs involves modulation of central dopamine function and the tone of cerebral blood vessels. mAChRs of the M₂ subtype mediate muscarinic agonist-induced bradycardia, tremor, hypothermia, and autoinhibition of release in several brain regions. M₄ mAChRs modulate dopamine activity in motor tracts and act as inhibitory autoreceptors in striatum. Thus, as elucidated by gene targeting technology, mAChRs have widespread and manifold functions in the periphery and brain.     

G Protein and β-Arrestin Signaling Bias at the Ghrelin Receptor

The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through G_q/11, G_i/o, and G_12/13 as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca²⁺ mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events.     

Characterization of Phospholipase C�� Enzymes with Gain-of-Function Mutations

Phospholipase Cγ isozymes (PLCγ1 and PLCγ2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLCγ2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLCγ1 and PLCγ2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLCγ2 without increasing Rac binding. Importantly, the activation of the ALI14-PLCγ2 and corresponding PLCγ1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.Phosphoinositide-specific phospholipase C (PLC)² enzymes, comprising several families (PLCβ, γ, δ, ϵ, η, and ζ), have been established as crucial signaling molecules involved in regulation of a variety of cellular functions (1–4). PLC-catalyzed formation of the second messengers, inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol, from phosphatidylinositol 4,5-bisphosphate (PIP₂), constitutes one of the major cell signaling responses. These second messengers provide a common link from highly specific receptors for hormones, neurotransmitters, antigens, and growth factors to downstream, intracellular targets; thus, they contribute to regulation of biological functions as diverse as cell motility, fertilization, and sensory transduction. Despite this central role for PLC enzymes in signaling networks, the molecular details of their regulation and possible subversion of these regulatory mechanisms in disease remain poorly understood.Of two PLCγ enzymes, PLCγ1 is ubiquitously expressed and appears to regulate a multitude of cellular functions in many tissues. Plcg1-null mice die by embryonic day 9, highlighting the widespread importance of this enzyme (5). PLCγ1 is activated in response to growth factor stimulation; in addition, its function in T-cell responses has been extensively documented (1). PLCγ2, in contrast, is most highly expressed in cells of the hematopoietic system and plays a key role in regulation of the immune response. Consistent with this, Plcg2-null mice display defects in the functioning of B cells, platelets, mast cells, and natural killer cells (6).Both PLCγ enzymes have also been implicated in signaling events underlying aberrant cellular responses. PLCγ1 is critically involved in the regulation of cancer cell motility (7–11) while PLCγ2 has been implicated in deregulation of the immune responses resembling Btk-dependent X-linked agammaglobulinaemia and SLE disease in humans (12–14). It has been suggested that, in cancer cells, PLCγ1 could function as a key, rate-limiting, common component involved in cell motility triggered by several growth factors and integrins (7). In some cancer cells, this increased motility could result from deregulation i.e. higher levels of expression of PLCγ1 (15, 16). The possibility that the activity of PLCγ could be up-regulated due to mutation has not yet been fully investigated in cancer. Previous studies of PLCγ2, however, have demonstrated the first gain-of-function mutation in a PLC molecule in the context of an organism, and shown that, in principle, PLC activity can be greatly enhanced by point mutations (13). Furthermore, this work has demonstrated that such a mutation is linked to a dramatic phenotypic disorder. By using a large scale ENU mutagenesis to discover new immune regulators, several mouse strains were generated with spontaneous autoimmune and inflammatory symptoms; two of these strains harbor a mutation in PLCγ2. In addition to the previously described ALI5 mutation (13) the ALI14 mutation has been identified very recently.³ Strikingly, the well-characterized ALI5 phenotype has shown that the mutation affects many cellular functions deregulated in Plcg2-null mice. Notably, while in Plcg2-null mice such responses are lacking, the ALI5 mutation resulted in their enhancement. In particular, further analyses of the ALI5 mutation in the context of signaling in B-cells have demonstrated that calcium responses to the crosslinking of the B-cell receptor were enhanced and prolonged resulting in enhanced deletion of B cells and autoreactivity (13).The domain organization of PLCγ enzymes is characterized by the insertion of a highly structured region (PLCγ-specific array, γSA) between the two halves of the TIM-barrel catalytic domain common to all PLCs. The γSA comprises a split PH (spPH) domain flanking two tandem SH2 domains and a SH3 domain (1). A distinct regulatory feature of PLCγ enzymes is that their activation is linked to an increase in phosphorylation of specific tyrosine residues (most notably within the γSA) by receptor and non-receptor tyrosine kinases (17, 18). Furthermore, multiple protein-protein interactions (mainly mediated by SH2 domains) also contribute to activation and have an important role in localizing PLCγ into protein complexes with different binding partners, depending on cell type and specific cellular compartments. One mode of activation that is specific for the PLCγ2 isozyme is direct binding to and activation by Rac. The interaction involves the spPH domain, and this activation mechanism does not require tyrosine phosphorylation (19, 20). In molecular terms, changes that lead to PLC activation in response to different input signals, or due to point mutations, are not well understood and require further studies.Here we describe further analysis of the two gain-of-function mutations, ALI5 and ALI14, obtained using ENU mutagenesis. These mutations map to different regions in PLCγ2, and we performed detailed analysis of these regions in both PLCγ isozymes. To characterize the molecular mechanism of gain-of-function, we combined studies in vitro and in different cellular signaling contexts. We have found that ALI5- and ALI14-type point mutations lead, by distinct mechanisms, to an enhancement of responses to a variety of input signals while their combination results in a constitutively active PLC enzyme.     

Role of α7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP^C) is a conserved glycosylphosphatidylinositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP^C extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrP^C-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP^C engagement induces an increase in intracellular Ca²⁺ levels. This effect was not detected in PrP^C-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP^C. Using a best candidate approach to test for potential channels involved in Ca²⁺ influx evoked by STI1-PrP^C, we found that α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrP^C-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP^C and allowed reconstitution of signaling by PrP^C-STI1 interaction. These results indicate that STI1 can interact with the PrP^C·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.     

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

The concept of “functional selectivity” or “biased signaling” suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the β₂-adrenergic receptor (β₂AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β₂AR FRET sensor. In this β₂AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard “full” agonist isoproterenol. Furthermore, norepinephrine-induced changes in the β₂AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial β₂AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G_s and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering β-arrestin2 recruitment to the cell surface and its interaction with β₂AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the β₂AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G_s and less efficient in signaling to β-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the β₂AR and link it to distinct conformational changes in the receptor.     

ERK5 Activation by Gq-Coupled Muscarinic Receptors Is Independent of Receptor Internalization and β-Arrestin Recruitment

G-protein-coupled receptors (GPCRs) are known to activate both G protein- and β-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK) pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII) failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.     

Pro-prion Binds Filamin A,Facilitating Its Interaction with Integrin β1, and Contributes to Melanomagenesis

Filamin A (FLNA) is an integrator of cell mechanics and signaling. The spreading and migration observed in FLNA sufficient A7 melanoma cells but not in the parental FLNA deficient M2 cells have been attributed to FLNA. In A7 and M2 cells, the normal prion (PrP) exists as pro-PrP, retaining its glycosylphosphatidyl-inositol (GPI) anchor peptide signal sequence (GPI-PSS). The GPI-PSS of PrP has a FLNA binding motif and binds FLNA. Reducing PrP expression in A7 cells alters the spatial distribution of FLNA and organization of actin and diminishes cell spreading and migration. Integrin β1 also binds FLNA. In A7 cells, FLNA, PrP, and integrin β1 exist as two independent, yet functionally linked, complexes; they are FLNA with PrP or FLNA with integrin β1. Reducing PrP expression in A7 cells decreases the amount of integrin β1 bound to FLNA. A PrP GPI-PSS synthetic peptide that crosses the cell membrane inhibits A7 cell spreading and migration. Thus, in A7 cells FLNA does not act alone; the binding of pro-PrP enhances association between FLNA and integrin β1, which then promotes cell spreading and migration. Pro-PrP is detected in melanoma in situ but not in melanocyte. Invasive melanoma has more pro-PrP. The binding of pro-PrP to FLNA, therefore, contributes to melanomagenesis.     

Interaction with caveolin-1 modulates G protein coupling of mouse β3-adrenoceptor

Caveolins act as scaffold proteins in multiprotein complexes and have been implicated in signaling by G protein-coupled receptors. Studies using knock-out mice suggest that β(3)-adrenoceptor (β(3)-AR) signaling is dependent on caveolin-1; however, it is not known whether caveolin-1 is associated with the β(3)-AR or solely with downstream signaling proteins. We have addressed this question by examining the impact of membrane rafts and caveolin-1 on the differential signaling of mouse β(3a)- and β(3b)-AR isoforms that diverge at the distal C terminus. Only the β(3b)-AR promotes pertussis toxin (PTX)-sensitive cAMP accumulation. When cells expressing the β(3a)-AR were treated with filipin III to disrupt membrane rafts or transfected with caveolin-1 siRNA, the cyclic AMP response to the β(3)-AR agonist CL316243 became PTX-sensitive, suggesting Gα(i/o) coupling. The β(3a)-AR C terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant β(3a)-ARs (F389A/Y392A/F398A or P384S/F389A) promoted PTX-sensitive cAMP responses, and in situ proximity assays demonstrated an association between caveolin-1 and the wild type β(3a)-AR but not the mutant receptors. In membrane preparations, the β(3b)-AR activated Gα(o) and mediated PTX-sensitive cAMP responses, whereas the β(3a)-AR did not activate Gα(i/o) proteins. The endogenous β(3a)-AR displayed Gα(i/o) coupling in brown adipocytes from caveolin-1 knock-out mice or in wild type adipocytes treated with filipin III. Our studies indicate that interaction of the β(3a)-AR with caveolin inhibits coupling to Gα(i/o) proteins and suggest that signaling is modulated by a raft-enriched complex containing the β(3a)-AR, caveolin-1, Gα(s), and adenylyl cyclase.     

Demonstration of a Direct Interaction between β2-Adrenergic Receptor and Insulin Receptor by BRET and Bioinformatics

Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and β₂-adrenergic receptor (β₂AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on β₂AR properties, we showed that IR coexpression had no effect on the ligand binding properties of β₂AR; however, IR reduced β₂AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between β₂AR and IR, we employed quantitative BRET² saturation and competition assays. Saturation assay data suggested constitutive β₂AR and IR homo- and heteromerization. Calculated acceptor/donor (AD₅₀) values as a measure of the relative affinity for homo- and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET² signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET² data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of β₂AR and IR identified intracellular domains encompassing residues at the end of the 7^th TM domain and C-terminal tail of β₂AR and a cytoplasmic part of the IR β chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order β₂AR:IR oligomer formation, likely comprising heteromers of homodimers.