**1. Introduction**

Brassinosteroids (BRs) are plant-specific steroid hormones and play essential roles in a broad range of plant growth and developmental processes, including cell elongation, cell division, and differentiation, seed germination, stomata formation, root development, vascular differentiation, plant architecture, flowering, male fertility, and senescence [1–4]. BRs are also involved in responding to various abiotic and biotic stresses, such as drought, flooding, salinity, extreme temperatures, microbial pathogens, and insect herbivores [5–7]. Plants with defects in BR biosynthesis or signaling show a characteristic set of developmental defects, including dwarfed statue, male sterility, delayed senescence and flowering, and photomorphogenesis in the dark [8,9].

Using various experimental and theoretical approaches, including genetics, biochemistry, cell biology, chemical biology, structural biology, proteomics, transcriptomics, genomics, mathematical modeling, and computational dynamics simulation, a series of important BR signaling components have been well established and intensively studied, revealing a protein phosphorylation-mediated BR signaling cascade [10–12] (Figure 1). BRs are perceived at the plasma membrane (PM) by the extracellular domains of BRI1 (Brassinosteroid-Insensitive1) receptor, a leucine-rich repeat receptor-like kinase (LRR-RLK) [9,13], and its co-receptor BAK1 (BRI1-Associated receptor Kinase1, also known as SERK3 for Somatic Embryogenesis Receptor Kinase3) [14–16], a versatile LRR-RLK involved in many

signaling processes [17]. BR binding to the BR-binding pocket formed by the extracellular domains of BRI1 and BAK1 is thought to trigger conformational changes of their cytoplasmic domains [18,19]. BRI1 subsequently phosphorylates its inhibitor BKI1 (BRI1 Kinase Inhibitor1) and induces its dissociation from the PM [14,18,20,21], thus enabling heterodimerization, reciprocal phosphorylation, and full activation of the kinase activities of BRI1 and BAK1 [14,18,20–23]. The fully activated BRI1 triggers a series of phosphorylation or dephosphorylation events to transduce the extracellular BR signals into the cytosol. BRI1 phosphorylates BSK1 (BR-Signaling Kinase1), CDG1 (Constitutive Differential Growth1), and some of their homologs, leading to phosphorylation and subsequent activation of members of a unique family of protein phosphatases with Kelch-like domain (PPKLs) that include BSU1 (bri1 suppressor1) and BSU1-Like1-3 (BSL1-3) [24–26]. It is generally believed that the phosphorylated BSU1/BSLs inactivate BIN2 (Brassinosteroid-Insensitive2), which is a member of the plant GSK3 (Glycogen Synthase Kinase3)-like kinase family, via dephosphorylation of a phosphorylated tyrosine (Tyr) residue in the activation loop of BIN2 [27]. The dephosphorylated BIN2 also interacts with KIB1 (Kink suppressed in *bzr1-1D*1), an F-box E3 ubiquitin ligase, leading to BIN2 ubiquitination and proteasome-mediated degradation [28]. Upon BIN2 inactivation and degradation, two highly similar BIN2 substrates, BZR1 (Brassinazole-resistant1) and BES1 (bri1-EMS suppressor1) [29,30] are rapidly dephosphorylated by certain nuclear-localized members of the PP2A (protein phosphatase 2A) family [31], leading to their nuclear accumulation. The dephosphorylated BZR1 and BES1 bind to their target promoters containing BRRE (BR-response element) (CGTGC/TG) and/or E-box (CANNTG) motif to regulate expression of thousands of BR-responsive genes that are crucial for plant growth and development [32,33].

**Figure 1.** A current model of BR signaling. When BRs (brassinosteroids) are absent (left), BRI1 (Brassinosteroid-Insensitive1) is kept inactive by its autoinhibitory C-terminus and BKI1 (BRI1 Kinase Inhibitor1) association. BIN2 (Brassinosteroid-Insensitive2) is constitutively active and phosphorylates BES1 (bri1-EMS suppressor1)/BZR1 (Brassinazole-resistant1) transcription factors to promote their degradation and 14-3-3-mediated cytosolic retention, and to directly inhibit their DNA-binding activities. When BRs are present (right) and bind to the extracellular domains of BRI1 and its co-receptor BAK1 (BRI1 Associated receptor Kinase1) to activate the two receptor kinases, leading to dissociation of BKI1 from BRI1, phosphorylation and activation of BSKs (BR-signaling kinases)/CDGs (Constitutive Differential Growth) and BSU1 (bri1 suppressor1). The activated BSU1 dephosphorylates and inactivates BIN2 while KIB1 (Kink suppressed in *bzr1-1D*1) promotes BIN2 degradation, causing nuclear accumulation of PP2A (protein phosphatase 2A)-dephosphorylated BES1/BZR1 that bind BRRE (BR response element)/E-box-containing promoters to regulate expression of thousands of BR-responsive genes important for plant growth and development.

In the past few years, several excellent reviews were published that summarized the significant progresses in understanding how the BR signal is perceived at the cell surface and how the extracellular BR signal is transduced into the nucleus to regulate a wide range of plant developmental and physiological processes [10–12,34]. In this review, we present a critical analysis of currently available data on three early BR signaling components, including the BR receptor BRI1, its coreceptor BAK1, and the crucial negative regulator BIN2, highlighting recent findings and discussing controversies and unanswered questions on the regulatory mechanisms that control their protein abundance, subcellular localizations, and signaling activities.

#### **2. BRI1, the BR Receptor**

BRI1 localizes to the PM and belongs to a large and plant-specific family of LRR-RLKs, which is composed of 223 members in *Arabidopsis thaliana* [35]. BRI1 consists of an extracellular LRR domain of 25 LRRs, a single-pass transmembrane segment, and a cytoplasmic kinase domain [9]. The 25 LRRs are interrupted by a 70-residue island domain (ID, from amino acid Lys<sup>586</sup> to Met657), which constitutes the BR binding domain with the 22nd LRR [36]. Its cytoplasmic kinase domain can be subdivided into the JM (juxtamembrane) region, a canonical serine/threonine (Ser/Thr) kinase domain, and a short C-terminal tail (CT) of 36 amino acids (AAs). The Arabidopsis genome encodes three BRI1 homologs: BRL1 (BRI1-Like1), BRL2 (also known as VH1 for Vascular Highway1 [37]), and BRL3. BRL1 and BRL3 can also bind BRs and function as BR receptors [38,39], whereas BRL2/VH1 does not bind BR but functions in a BR-independent manner to regulate vascular development [37,40]. Gene expression analysis and genetic studies revealed that BRI1 is expressed in most plant tissues/organs, whereas BRLs are found only in the vascular tissues and stem cell niches [41]. BRLs have been shown to be involved in vascular development [42] and plant tolerance to abiotic stresses, such as hypoxia and drought [43,44].

#### *2.1. Maintaining the Inactive State in the Absence of BR*

In the absence of BR ligands, BRI1 is maintained at its inactive state via its inhibitory CT and its binding to BKI1 at the PM [20,23]. Removal of the BRI1 CT not only increased the in vitro kinase activity of a recombinant BRI1 kinase protein, but also led to a hyperactive BR receptor in vivo [23]. Further investigation will likely be required to fully understand the mechanism of this autoinhibitory activity of the BRI1 kinase activity given the missing CT in several available crystal structures of the BRI1 kinase domain [45,46]. The 36-AA CT could block the substrate binding site to interfere with the in vitro homodimerization for the auto(trans)phosphorylation activity or the in vivo binding of the kinase domains of BRI1 and BAK1. In addition to the "*cis*" autoinhibition, the binding of the PM-associated BKI1 to the BRI1 cytoplasmic domain demonstrates a "*trans*" inhibitory mechanism to prevent association and subsequent cross phosphorylation of the kinase domains of BRI1 and BAK1, which is likely triggered by BR-independent heterodimerization of their extracellular domains known to occur in plant cells [15,47,48]. A previous genetic demonstration of the absolute requirement of BAK1/SERKs for the BRI1's activation [49] implies that BRI1 can be kept in its inactive state via competitive binding of BAK1 with certain BAK1-interacting proteins. Indeed, recent studies revealed that ligand-independent interaction of BAK1 with BIR3 (BAK1-Interacting Receptor-like kinase3) could inhibit BR-stimulated BAK1-BRI1 heterodimerization and BRI1 activation (see below for more discussion on the BAK1-BIR interaction) [50].

### *2.2. BRI1 Activation*

It was well known that many kinases are activated via phosphorylation of crucial Ser/Thr residues in the activation segmen<sup>t</sup> catalyzed by upstream kinases [51]. In animal cells, many receptor kinases are activated by homodimerization and auto(trans)phosphorylation [52,53]. Despite several reports of BRI1 homodimerization in vitro or in vivo [15,23,54,55], BRI1 activation requires its heterodimerization and subsequent transphosphorylation with its coreceptor BAK1/SERKs. An earlier in vitro kinase assay with dephosphorylated recombinant kinases of BRI1 and BAK1 showed that neither BRI1 nor BAK1 was active when incubated alone with the ATP-containing kinase reaction bu ffer but became active when incubated together [45]. Similarly, the full-length BRI1 or BAK1 was inactive when expressed alone in yeas<sup>t</sup> cells but became active kinases when they were coexpressed. Neither BRI1 nor BAK1 was active when coexpressed in yeas<sup>t</sup> with a kinase-dead partner [18]. More important, a BRI1-FLAG fusion protein could not be activated by exogenously applied BR in a transgenic Arabidopsis line that lacks BAK1 and its two close homologs [49]. Furthermore, a recent study showed that ligand-independent heterodimerization of a chimeric LRR-RLK, which was composed of the extracellular domain of BIR3 and the intracellular domain of BRI1, and BAK1 led to constitutive activation of the BR signaling pathway [56]. All these biochemical and genetic/transgenic experiments argue against the widely accepted sequential-phosphorylation model. This model posits that BR binding-triggered conformational changes result in weak activation of BRI1 and its subsequent phosphorylation and activation of BAK1, which can then transphosphorylate BRI1 for its full activation [21]. Further studies, especially structural analysis of the ligand-bound full-length BRI1-BAK1 complexes and mass spectrometry (MS)-based quantitative phosphorylation analysis of BRI1 in *bak1*/*serk* mutants, are needed to fully understand the activation mechanism of BRI1 and the genetic requirement of BAK1/SERKs for its activation.

Structural analyses of the BRI1-BAK1 extracellular heterodimers and molecular dynamics simulations suggested that BR binding to the extracellular domain of BRI1 causes conformational changes in BRI1 [57,58]. Such subtle structural changes not only stabilize the ID to create a docking platform for the BRI1-BAK1 association, which is further stabilized by a bound active BR (functioning as "molecular glue" to interact with the N-terminal cap of BAK1), but also potentially create a secondary interface involving the N-terminal 12 LRRs of BRI1 to interact with BAK1 [16,19] (Mo ffett and Shukla, 2020 bioRxiv: http://doi.org/10.1101/630640). Despite lack of experimental evidence or structural information, it has been widely accepted that stable association of the extracellular domains of BRI1 and BAK1 brings their cytoplasmic domains into close proximity, thus permitting cross phosphorylation, especially at Ser<sup>1044</sup> of BRI1 and Thr<sup>450</sup> of BAK1 within their respective activation loop, and subsequent activation of both kinases [45,46,59]. A recent structural modeling study suggested that the cytoplasmic domains of BRI1 and BAK1 could weakly interact independently of BR binding to their extracellular domains [60]. However, it is important to point out that the structural models of the kinase domains of BRI1 and BAK1 used for the modeling study were derived from autophosphorylated and activated kinase domains with stabilized αC-helixes and activation segments.

Structural models of the activated BRI1 kinase domain [45,46] suggested that the phosphorylated Ser<sup>1044</sup> is likely trapped in a positively-charged phosphate-binding pocket consisting of Arg<sup>922</sup> (at the beginning of the αC-helix), Arg<sup>1008</sup> [within the HRD (His-Arg-Asp) motif], and Arg<sup>1032</sup> (at the beginning of the β9-strand), thus stabilizing the activation loop and establishing an active kinase conformation (Figure 2). Specifically, the interaction of the phosphorylated Ser<sup>1044</sup> (pSer1044) and Arg<sup>922</sup> of the αC-helix is likely critical for creating the so-called regulatory-spine (R-spine) consisting of 5 residues [the anchoring Asp<sup>1068</sup> residue of the αF-helix, His<sup>1007</sup> of the HRD motif, Phe<sup>1028</sup> of the DFG (Asp-Phe-Gly) motif, Ile<sup>931</sup> of the αC-helix, and Leu<sup>942</sup> of the β4-strand] (Figure 2). The assembly of the R-spine has been considered as a crucial indicator of an active conformation of eukaryotic protein kinases [61,62]. As expected, a Ser1044-Ala mutation resulted in a strong loss of kinase and signaling activity of BRI1 in vitro and in vivo [22].

**Figure 2.** A structural model of the activated BRI1 kinase domain. Shown here are three (0◦, 60◦, and 180◦) rotational views of a rainbow-colored ribbon model of the crystal structure of the BRI1 kinase domain (Protein Data Bank No. 4oh4). Individual α-helices (αB-αI) and β-strands (β1-β5) were labelled. The magenta-colored sticks indicate phosphorylated Ser/Thr residues with the magenta dots surrounding Ser<sup>1044</sup> and Thr<sup>1049</sup> of the activation segment, the green sticks denote the Lys<sup>911</sup> and Glu<sup>927</sup> residues that form the salt bridge between the β3-strand and αC-helix, the red sticks mark the three positively charged residues of the phosphate-binding pocket, and the orange sticks represent the phosphorylated Tyr residue. The red spheres show adenylyl-imidodiphosphate (a non-hydrolysable ATP analog), the orange spheres indicate the gatekeeper Tyr<sup>956</sup> residue that is also phosphorylated in in vitro assays, and the light-blue spheres denote the five regulatory-spine (R-spine) residues (from lower to upper: Asp1068, His1077, Phe1028, Ile931, and Leu942). The rainbow bar indicates the order of amino acids (AAs) from the N-terminus (blue) to the C-terminus (red).

In addition to the pSer<sup>1044</sup> residue within the activation loop, previous studies revealed an essential role of a conserved Ser/Thr residue (Thr1049) in the "P+1 loop" [21,22], which is thought to be the docking site for the backbone of the substrate P-site and the side-chain of the P+1 residue [61]. Previous bioinformatic analysis indicated that this position is occupied by a Ser/Thr residue in >99% of the so-called "RD"-type RLKs containing Arg-Asp residues within the conserved HRD motif [21]. By contrast, only 30% of "non-RD"-type RLKs contain a Ser/Thr residue at this position. Mutating Thr<sup>1049</sup> to Ala completely inhibited the in vitro phosphorylation activity of a recombinant BRI1 kinase domain and greatly reduced its signaling activity in transgenic Arabidopsis plants [23], demonstrating its crucial role in BRI1 kinase activity. Mutating the equivalent Ser/Thr residue in several other "RD"-type LRR-RLKs resulted in loss of in vitro phosphorylation activity [63]. Further studies will be needed to determine whether the phosphorylation of this residue is truly required for BRI1 activation as the phosphorylation-mimic Thr1049-Asp mutation also led to a strong loss of the in vitro phosphorylation activity of a recombinant BRI1 kinase [21]. Based on the structural model (Figure 2), the phosphorylation of this residue could potentially interfere with ATP binding and/or substrate binding.

The activated BRI1 is thought to autophosphorylate additional Ser, Thr, and Tyr residues in both N-lobe and C-lobe (Figure 2). The phosphorylated N-lobe residues include Ser838, Thr842, Thr846, Thr851, and Ser<sup>858</sup> of the JM (not shown in Figure 2), Thr<sup>872</sup> and Thr<sup>880</sup> on the αB-helix that caps the N-lobe, Ser<sup>887</sup> and Ser<sup>891</sup> on both ends of the β1-strand, Ser<sup>906</sup> in the β2-β3 loop, Ser<sup>917</sup> in the β3-αC loop (missing in the BRI1 crystal structure), and Thr<sup>930</sup> on the αC-helix. The phosphorylated C-lobe residues include Ser<sup>963</sup> at the start of the αD-helix, Ser981, Thr982, and Ser<sup>990</sup> of the αE-helix, Ser<sup>1012</sup> and Ser<sup>1013</sup> of a half helical twist before the β7-strand, Ser<sup>1026</sup> located between the β8-strand and the DFG motif, Thr1039, Ser1042, and Thr<sup>1045</sup> of the activation segment, Ser<sup>1071</sup> and Thr<sup>1081</sup> of the αF-helix, Ser<sup>1109</sup> located at the start of the twisted helical αG-αH linker, Thr<sup>1147</sup> at the start of the αI-helix, and at least 5 residues in the CT (Ser1166, Ser1168, Thr1169, Ser1179, and Ser1187) [21,22,63–66] (Figure 2). Among

those phosphorylated residues, at least 11 were identified from immunoprecipitated BRI1 fusion protein from transgenic Arabidopsis plants, including 6 uniquely identified (Ser838, Ser858, Thr872, Thr880, Thr982, and Ser1168), 2 additional Thr sites (most likely Thr<sup>842</sup> and Thr846) and 3 ambiguous sites within the activation fragment [22]. In addition, at least 3 Tyr residues were reported to be autophosphorylated, including Tyr<sup>831</sup> (in the JM), Tyr<sup>956</sup> (the gatekeeper residue on the β5-strand), and Tyr<sup>1072</sup> (on the αF-helix) [67,68] (Figure 2). It should be noted that the published MS data from several in vitro and in vivo phosphorylation site-mapping experiments of BRI1 did not identify any phosphorylated Tyr residue [21,22,63–66]. The 3 phosphorylated Tyr residues (Tyr831, Tyr956, and Tyr1072) that were discussed in the current literature were deduced from immunoblot assays with generic and site-specific anti-phosphorylated Tyr (anti-pTyr) antibodies [67,68]. It is generally believed that these phosphorylated Ser/Thr/Tyr sites could regulate the abundance and/or signaling activity of BRI1 and create docking sites for binding downstream signaling components or regulators to build a BRI1 signaling complex, which likely include sca ffolding proteins such as TTLs (Tetratricopeptide Thioredoxin-Like proteins) and BSK3 [69,70]. The activated BRI1 can then transphosphorylate these regulators and downstream targets to transduce the extracellular BR signal into the cytosol, ultimately reaching the nucleus to alter gene expression.

One question that remains to be answered is how binding of BR to BRI1 triggers phosphorylation of BKI1. As discussed above, the activation of BRI1's kinase activity requires its heterodimerization and transphosphorylation with BAK1, a process that is prevented by the PM-localized BRI1-binding BKI1, while BKI1 dissociation from BRI1 and the PM absolutely requires phosphorylation by activated BRI1 [71,72]. Previous studies showed that BRI1-catalyzed Tyr phosphorylation at the conserved Tyr211, which likely requires BRI1-catalyzed phosphorylation at Ser<sup>270</sup> and Ser<sup>274</sup> [72], is necessary and su fficient for its dissociation from the PM [71]. It might be possible that BR binding to the BRI1's extracellular domain triggers ye<sup>t</sup> undefined conformational changes in its cytoplasmic domain. Such structural changes could weaken the BRI1-BKI1 binding and permit weak BRI1-BAK1 association to allow the BAK1-catalyzed transphosphorylation of Thr<sup>1044</sup> in the activation loop of BRI1, leading to further conformational changes and full activation of BRI1. Activated BRI1 can subsequently phosphorylate BKI1 at Ser270/<sup>274</sup> and Tyr211, resulting in BKI1 dissociation from BRI1 and the PM and a stronger BRI1-BAK1 binding. A better understanding of the BRI1-BKI1 and BRI1-BAK1 interaction requires further structural analyses of the protein complexes of full-length proteins in the absence or presence of active BRs.

#### *2.3. Attenuation and Deactivation*

The magnitude and duration of the BR signaling is dynamically regulated by activation and deactivation of the BR receptor. One simple mechanism of receptor inactivation or attenuation is mediated by autophosphorylation. Previous studies suggested that autophosphorylation at certain residues inhibited the in vitro kinase activity of an *E. coli* expressed BRI1 kinase domain and reduced the physiological activity of BRI1 in transgenic Arabidopsis plants [21,23,67,73]. For example, mutating Thr<sup>872</sup> to Ala, which is located at the start of the αB-helix that caps the N-lobe of 5 antiparallel β-strands (Figure 2), could significantly enhance the in vitro autophosphorylation activity or transphosphorylation activity towards an artificial peptide substrate [22]. It is interesting to note that Thr<sup>872</sup> and Thr880, known to be phosphorylated and located at the start and end of the αB-helix (Figure 2), are highly conserved among 213 Arabidopsis LRR-RLK [22], suggesting that phosphorylation of these residues might be a conserved attenuation mechanism for many plant LRR-RLKs. Similarly, Ser891, located in the β1-β2 ATP-binding loop (better known as "glycine-rich loop" or "G-loop") containing the GXGXXG-motif (X indicating any AA), was previously shown to deactivate BRI1 [73], likely by interfering with ATP binding, as the "G-loop" is known for positioning the three phosphate groups and the adenine ring of ATP [61]. An earlier study also suggested that phosphorylation of two tyrosine residues, Tyr<sup>831</sup> (located in the JM) and Tyr<sup>956</sup> (a critical gatekeeper of the ATP binding pocket), might also inhibit the BRI1 kinase and signaling activity [67]. However, the lack of appropriate phosphorylation-mimic AA for a pTyr residue makes it complicated to interpret the published experimental results on the regulatory roles of BRI1's Tyr phosphorylation.

In addition to the "*cis*" attenuation by autophosphorylation, the signaling activity of BRI1 could be "*trans*" attenuated by dephosphorylation of certain phosphorylated Ser/Thr residues through PP2A [66,74]. A PP2A holoenzyme is composed of three subunits: a catalytic C subunit, a regulatory B subunit, and a sca ffolding A subunit. A B subunit from one of the 3 subfamilies in Arabidopsis: B, B', and B", determines the substrate specificity, while an A subunit brings the B and C subunits together to form an active protein phosphatase complex [75]. An earlier study implicated the Arabidopsis SBI1 (suppressor of bri1), a leucine carboxylmethyltransferase, in methylating the C subunits of PP2A to enhance the PM-association of certain PP2A holoenzymes [74]. As a result, PP2A could bind and dephosphorylate activated BRI1, leading to increased BRI1 degradation and attenuated BR signaling. Further support for the involvement of PP2A in attenuating BR signaling came from a recent study showing that at least four cytoplasm-localized PP2A B' subunits, including B' γ, B'η, B'θ, and B'ζ [75], interact with BRI1 and mediate its dephosphorylation and inactivation [66]. Overexpression of any of these *PP2A-B'* genes in a weak *bri1-5* mutant significantly enhanced its dwarf phenotype and further reduced its BR signaling activity. By contrast, simultaneously eliminating these four *PP2A-B'* genes could enhance BR signaling and partially suppressed the dwarf phenotype of a weak BR-deficient mutant *det2* (*de-etiolation2*) [66]. Quantitative MS assays of autophosphorylated BRI1 kinase domain incubated with immunoprecipitated PP2A complexes identified several autophosphorylated Ser/Thr residues as the potential dephosphorylation sites of PP2A, including a phosphorylated residue (Thr872, Thr880, or Ser<sup>887</sup> on the αB-helix and the αB-β1 loop), Ser<sup>917</sup> (in the β3- αC loop), Ser<sup>981</sup> (on the αD helix), and four phosphorylated Ser/Thr residues in the CT (Ser1166, Ser1168, Thr1169, or Ser1172, and S<sup>1179</sup>/Thr1180) [66]. An earlier mutagenesis experiment indicated that individual mutations of these Ser/Thr residues (except Thr872) had little impact on the in vitro autophosphorylation activity of the BRI1 kinase but did reduce its transphosphorylation activity towards a peptide substrate [22]. In addition, the dephosphorylated CT was previously shown to exert inhibitory e ffects on BRI1 signaling activity [23]. Further MS analyses of the endogenous BRI1 in *PP2A B'*-overexpressing transgenic Arabidopsis lines and the Arabidopsis quadruple mutant lacking B' γ, B'η, B'θ, and B'ζ are needed to pinpoint the exact Ser/Thr residues that are dephosphorylated by PP2A. Given the importance of Tyr phosphorylation in BR signaling [67,68,71] and a previous report of Tyr dephosphorylation of an LRR-RLK immunity receptor EFR (Elongation Factor-Tu Receptor) by a bacterial tyrosine phosphatase [76], it will be interesting to investigate if the BRI1 signaling activity could also be regulated by a member of the Arabidopsis PTP/DSPP (protein tyrosine phosphatase/dual specificity protein phosphatase) family [77].

#### *2.4. Regulating the Abundance of BRI1 on the PM*

#### 2.4.1. Tra fficking from the ER to the PM

The BR signaling activity at the PM is also controlled by the amount of the PM-localized BRI1 receptor, which starts its secretory journey in the endoplasmic reticulum (ER) (Figure 3). It was well known that the ER houses several stringent quality control (QC) systems that permit export of only correctly folded proteins into the Golgi apparatus but retain incompletely or incorrectly folded proteins in the ER for chaperone-assisted folding repair or degradation via ER-associated degradation mechanism (ERAD) [78,79]. Two structurally defect but biochemically competent mutant variants of BRI1, bri1-5 with Cys69-Tyr mutation and bri1-9 carrying a Ser662-Phe mutation, are retained in the ER and degraded by ERAD [80–82] (Figure 3). Loss-of-function mutations in the ER quality control (ERQC) or ERAD system resulted in increased amounts of mutant bri1 receptors on the PM, thus partially suppressing the dwarfism phenotype of these *bri1* mutants as the corresponding mutant BRI1 proteins still retain partial signaling activity after being correctly targeted to the PM [83–86]. A recent study also suggested the presence of a ye<sup>t</sup> to be defined QC system at the PM to remove

misfolded/damaged PM-localized proteins, such as bri1-301 (with a Gly989-Ile mutation) that escapes from ERQC/ERAD [87,88]. TWD1 (Twisted Dwarf1), a well-studied cochaperone protein that was previously shown to be localized in the ER and at the PM, where it regulates auxin transport [89], also interacts with both BRI1 and BAK1 to enhance BR signaling [90,91]. Given its demonstrated interaction with HSP90 (heat shock protein 90) [92,93], it is tempting to speculate that TWD1 could promote optimal folding of BRI1 and its coreceptor, thus maximizing their BR signaling activities at the PM.

**Figure 3.** A current model of regulating the BRI1 abundance on the plasma membrane (PM)**.** Newly synthesized BRI1 is translocated through the Sec61 translocon from the ER (endoplasmic reticulum)-associated ribosomes into the ER where it undergoes chaperone-assisted protein folding. Correctly folded BRI1 is exported into the Golgi to continue its secretory journey through the TGN (*trans*-Golgi network)/EE (early endosome) to be targeted on the PM (1), whereas the incorrectly/misfolded BRI1, such as its mutant variants bri1-5 and bri1-9, are retained in the ER by ERQC (ER quality control) for refolding and degradation via ERAD (ER-associated degradation) that involves retrotranslocation and cytosolic proteasome. The PM-localized BRI1 or BRI1/BAK1 heterodimer is presumably ubiquitinated by the PUB12/13 (plant U-box protein12/13) E3 ligases and undergoes constitutive CME (clathrin-mediated endocytosis)-mediated internalization (2) or ligand-induced CIE (clathrin-independent endocytosis)-mediated internalization (3). The endocytosed BRI1 at the TGN/EE could be packaged into ILVs (intraluminal vesicles) and delivered to the vacuole for degradation via ESCRT (endosomal sorting complex required for transport)-mediated biogenesis of MVBs (multivesicular bodies) and eventual MVB-vacuole fusion (4). Alternatively, the TGN/EE-localized BRI1 can be recycled back to the PM via retromer-mediated cargo selection, microtubule-assisted vesicle trafficking, and exocyst/SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor)-involved exocytosis (5). The circled numbers indicate different secretion/trafficking routes. SNX1 (sorting nexin1) is one of the core retromer subunits, CLASP (cytoplasmic linker-associated protein) is a microtubule-associated protein, and ALIX (the Arabidopsis homolog of apoptosis-linked gene 2-interacting protein X) is a cytosolic ESCRT-associated protein.

#### 2.4.2. Targeting to Unique PM Nano/Microdomains

Recent fluorescence microscopy studies coupled with live-cell imaging techniques have revealed that plant LRR-RLKs, including BRI1 and BAK1, are not uniformly distributed on the PM but are

rather localized to specific PM subcompartments known as lipid rafts, nanodomains, or microdomains with unique lipid and protein compositions [48,94,95]. It was thought that members of the two protein families: Flotillins (Flot) and plant-specific remorins, might function as organization centers to form protein nanoclusters that are important for receptor signaling [55,95]. Interestingly, AtFlot1, one of the two Arabidopsis Flot homologs, was shown to coexist with BRI1 in a PM microdomain to influence BRI1 endocytosis [55] while OsREM4.1 (*Oryza sativa* remorin4.1), a member of the rice remorin family, interacted with OsSERK1, a rice homolog of BAK1, to regulate the heterodimerization of OsSERK1 with a rice homolog of BRI1, OsBRI1 [96]. It remains to be determined how AtFlot1 and OsREM4.1 recruit BRI1/OsBRI1 to unique nano/microdomains on the PM given the presence of >600 RLKs, but only 2 Flots and 16 remorins in Arabidopsis [97,98].
