Translocation expands the scope of the large clostridial toxin family.

Large clostridial toxins (LCTs) are a family of six homologous disease-causing proteins characterised by their large size (>200 kDa) and conserved multidomain architectures. Using their central translocation and receptor-binding domain (T domain), LCTs bind host cell receptors and translocate their upstream glycosyltransferase and cysteine protease domain across the endosomal membrane and into the cytosol. The recent discovery of hundreds of LCT-like T domains in diverse genomic contexts and domain architectures from bacteria other than clostridia has provided significant new insights into the enigmatic process of LCT translocation, but also has put the definition of what constitutes an LCT into question. In this opinion article, we discuss how these findings have expanded our understanding of LCT translocation and reshaped the scope of the LCT family.

Insulin-Activated Signaling Pathway and GLUT4 Membrane Translocation in hiPSC-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a cell model now widely used to investigate pathophysiological features of cardiac tissue. Given the invaluable contribution hiPSC-CM could make for studies on cardio-metabolic disorders by defining a postnatal metabolic phenotype, our work herein focused on monitoring the insulin response in CM derived from the hiPSC line UKBi015-B. Western blot analysis on total cell lysates obtained from hiPSC-CM showed increased phosphorylation of both AKT and AS160 following insulin treatment, but failed to highlight any changes in the expression dynamics of the glucose transporter GLUT4. By contrast, the Western blot analysis of membrane fractions, rather than total lysates, revealed insulin-induced plasma membrane translocation of GLUT4, which is known to also occur in postnatal CM. Thus, these findings suggest that hiPSC-derived CMs exhibit an insulin response reminiscent to that of adult CMs regarding intracellular signaling and GLUT4 translocation to the plasma membrane, representing a suitable cellular model in the cardio-metabolic research field. Moreover, our studies also demonstrate the relevance of analyzing membrane fractions rather than total lysates in order to monitor GLUT4 dynamics in response to metabolic regulators in hiPSC-CMs.

Cell-Penetrating Peptides for Use in Development of Transgenic Plants.

Genetically modified plants and crops can contribute to remarkable increase in global food supply, with improved yield and resistance to plant diseases or insect pests. The development of biotechnology introducing exogenous nucleic acids in transgenic plants is important for plant health management. Different genetic engineering methods for DNA delivery, such as biolistic methods, Agrobacterium tumefaciens-mediated transformation, and other physicochemical methods have been developed to improve translocation across the plasma membrane and cell wall in plants. Recently, the peptide-based gene delivery system, mediated by cell-penetrating peptides (CPPs), has been regarded as a promising non-viral tool for efficient and stable gene transfection into both animal and plant cells. CPPs are short peptides with diverse sequences and functionalities, capable of agitating plasma membrane and entering cells. Here, we highlight recent research and ideas on diverse types of CPPs, which have been applied in DNA delivery in plants. Various basic, amphipathic, cyclic, and branched CPPs were designed, and modifications of functional groups were performed to enhance DNA interaction and stabilization in transgenesis. CPPs were able to carry cargoes in either a covalent or noncovalent manner and to internalize CPP/cargo complexes into cells by either direct membrane translocation or endocytosis. Importantly, subcellular targets of CPP-mediated nucleic acid delivery were reviewed. CPPs offer transfection strategies and influence transgene expression at subcellular localizations, such as in plastids, mitochondria, and the nucleus. In summary, the technology of CPP-mediated gene delivery provides a potent and useful tool to genetically modified plants and crops of the future.

Bidirectional transfer of homeoprotein EN2 across the plasma membrane requires PIP2.

Homeoproteins are a class of transcription factors sharing the unexpected property of intercellular trafficking that confers to homeoproteins a paracrine mode of action. Homeoprotein paracrine action participates in the control of patterning processes, including axonal guidance, brain plasticity and boundary formation. Internalization and secretion, the two steps of intercellular transfer, rely on unconventional mechanisms, but the cellular mechanisms at stake still need to be fully characterized. Thanks to the design of new quantitative and sensitive assays dedicated to the study of homeoprotein transfer within HeLa cells in culture, we demonstrate a core role of phosphatidylinositol (4,5)-bisphosphate (PIP2) together with cholesterol in the translocation of the homeobox protein engrailed-2 (EN2) across the plasma membrane. By using drug and enzyme treatments, we show that both secretion and internalization are regulated according to PIP2 levels. The requirement for PIP2 and cholesterol in EN2 trafficking correlates with their selective affinity for this protein in artificial bilayers, which is drastically decreased in a paracrine-deficient mutant of EN2. We propose that the bidirectional plasma membrane translocation events that occur during homeoprotein secretion and internalization are parts of a common process.

Phosphorylation of cPLA2α at Ser505 Is Necessary for Its Translocation to PtdInsP2-Enriched Membranes.

Group IVA cytosolic phospholipase A2α (cPLA2α) is a key enzyme in physiology and pathophysiology because it constitutes a rate-limiting step in the pathway for the generation of pro- and anti-inflammatory eicosanoid lipid mediators. cPLA2α activity is tightly regulated by multiple factors, including the intracellular Ca2+ concentration, phosphorylation reactions, and cellular phosphatidylinositol (4,5) bisphosphate levels (PtdInsP2). In the present work, we demonstrate that phosphorylation of the enzyme at Ser505 is an important step for the translocation of the enzyme to PtdInsP2-enriched membranes in human cells. Constructs of eGFP-cPLA2 mutated in Ser505 to Ala (S505A) exhibit a delayed translocation in response to elevated intracellular Ca2+, and also in response to increases in intracellular PtdInsP2 levels. Conversely, translocation of a phosphorylation mimic mutant (S505E) is fully observed in response to cellular increases in PtdInsP2 levels. Collectively, these results suggest that phosphorylation of cPLA2α at Ser505 is necessary for the enzyme to translocate to internal membranes and mobilize arachidonic acid for eicosanoid synthesis.

Inhibition of MLKL-dependent necroptosis via downregulating interleukin-1R1 contributes to neuroprotection of hypoxic preconditioning in transient global cerebral ischemic rats.

BACKGROUND: Our previous study indicated that hypoxic preconditioning reduced receptor interacting protein (RIP) 3-mediated necroptotic neuronal death in hippocampal CA1 of adult rats after transient global cerebral ischemia (tGCI). Although mixed lineage kinase domain-like (MLKL) has emerged as a crucial molecule for necroptosis induction downstream of RIP3, how MLKL executes necroptosis is not yet well understood. In this study, we aim to elucidate the molecular mechanism underlying hypoxic preconditioning that inactivates MLKL-dependent neuronal necroptosis after tGCI. METHODS: Transient global cerebral ischemia was induced by the four-vessel occlusion method. Twenty-four hours before ischemia, rats were exposed to systemic hypoxia with 8% O2 for 30 min. Western blotting was used to detect the expression of MLKL and interleukin-1 type 1 receptor (IL-1R1) in CA1. Immunoprecipitation was used to assess the interactions among IL-1R1, RIP3, and phosphorylated MLKL (p-MLKL). The concentration of intracellular free calcium ion (Ca2+) was measured using Fluo-4 AM. Silencing and overexpression studies were used to study the role of p-MLKL in tGCI-induced neuronal death. RESULTS: Hypoxic preconditioning decreased the phosphorylation of MLKL both in neurons and microglia of CA1 after tGCI. The knockdown of MLKL with siRNA decreased the expression of p-MLKL and exerted neuroprotective effects after tGCI, whereas treatment with lentiviral delivery of MLKL showed opposite results. Mechanistically, hypoxic preconditioning or MLKL siRNA attenuated the RIP3-p-MLKL interaction, reduced the plasma membrane translocation of p-MLKL, and blocked Ca2+ influx after tGCI. Furthermore, hypoxic preconditioning downregulated the expression of IL-1R1 in CA1 after tGCI. Additionally, neutralizing IL-1R1 with its antagonist disrupted the interaction between IL-1R1 and the necrosome, attenuated the expression and the plasma membrane translocation of p-MLKL, thus alleviating neuronal death after tGCI. CONCLUSIONS: These data support that the inhibition of MLKL-dependent neuronal necroptosis through downregulating IL-1R1 contributes to neuroprotection of hypoxic preconditioning against tGCI.

Subcellular trafficking of tubular MDM2 implicates in acute kidney injury to chronic kidney disease transition during multiple low-dose cisplatin exposure.

Acute kidney injury (AKI) is the leading cause of renal failure, and quite a few patients will advance to chronic kidney disease (CKD) in the long term. Here, we explore the roles and mechanisms of tubular epithelial cells (TECs) during repeated cisplatin (CP) induced AKI to CKD transition (AKI-CKD). Previously, we reported that murine double minute 2 (MDM2), an E3-ubiquitin ligase, is involved in tubulointerstitial fibrosis. However, whether tubular MDM2 is implicated in AKI-CKD is undefined. Currently, we confirmed that during AKI-CKD, MDM2 shifts from nucleus to cell membrane in TECs both in vivo and in vitro. Whereas regulating MDM2 distribution chemically or genetically has a prominent impact on tubular disorders. And then we investigated the mechanisms of the above findings. First, in the nucleus, repeated CP administration leads to MDM2 reduction with escalated p53 and cell cycle G2/M arrest. On the other hand, multiple CP treatment increases the level of membranous MDM2 with ensuing integrin ß8 degradation and TGF-ß1 activation. More interestingly, anchoring MDM2 on cell membranes can mimic the reduction of integrin ß8 arousing by repeated CP exposure. Collectively, our findings provided the evidence that tubular MDM2 subcellular shuttling is involved in AKI-CKD through p53-G2/M arrest and integrin ß8 mediated TGF-ß1 activation.

Membrane anchoring of a curvature-inducing peptide, EpN18, promotes membrane translocation of octaarginine.

EpN18 is a curvature-inducing peptide, which loosens lipid packing upon interaction with the cell membrane, and facilitates cell-membrane penetration by arginine-rich cell-penetrating peptides, including octaarginine (R8). In the present study, we conjugated the N-terminal of EpN18 with a pyrenebutyryl (pBu) moiety, which acts as an anchoring unit that increases membrane interactions. Enhanced lipid-packing loosening and cytosolic translocation of R8 were observed by the pBu anchoring of EpN18.

Large-Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca*.

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter-pair-based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate-limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times, in agreement with the liposome study. Applied-field and steered molecular dynamics simulations added an atomistic view of the permeation events. It can be concluded that a concentration gradient of 1 µm Ptm leads to a translocation rate of about one molecule per second and per channel.

From TOM to the TIM23 complex - handing over of a precursor.

Mitochondrial precursor proteins with amino-terminal presequences are imported via the presequence pathway, utilizing the TIM23 complex for inner membrane translocation. Initially, the precursors pass the outer membrane through the TOM complex and are handed over to the TIM23 complex where they are sorted into the inner membrane or translocated into the matrix. This handover process depends on the receptor proteins at the inner membrane, Tim50 and Tim23, which are critical for efficient import. In this review, we summarize key findings that shaped the current concepts of protein translocation along the presequence import pathway, with a particular focus on the precursor handover process from TOM to the TIM23 complex. In addition, we discuss functions of the human TIM23 pathway and the recently uncovered pathogenic mutations in TIM50.

Evolution of mitochondrial protein import - lessons from trypanosomes.

The evolution of mitochondrial protein import and the systems that mediate it marks the boundary between the endosymbiotic ancestor of mitochondria and a true organelle that is under the control of the nucleus. Protein import has been studied in great detail in Saccharomyces cerevisiae. More recently, it has also been extensively investigated in the parasitic protozoan Trypanosoma brucei, making it arguably the second best studied system. A comparative analysis of the protein import complexes of yeast and trypanosomes is provided. Together with data from other systems, this allows to reconstruct the ancestral features of import complexes that were present in the last eukaryotic common ancestor (LECA) and to identify which subunits were added later in evolution. How these data can be translated into plausible scenarios is discussed, providing insights into the evolution of (i) outer membrane protein import receptors, (ii) proteins involved in biogenesis of α-helically anchored outer membrane proteins, and (iii) of the intermembrane space import and assembly system. Finally, it is shown that the unusual presequence-associated import motor of trypanosomes suggests a scenario of how the two ancestral inner membrane protein translocases present in LECA evolved into the single bifunctional one found in extant trypanosomes.

Annexin A2 acts as an adherent molecule under the regulation of steroids during embryo implantation.

We previously showed that annexin A2 (Axna2) was transiently expressed at the embryo-uterine luminal epithelium interface during the window of implantation and was involved in mouse embryo implantation. At the same time, Axna2 was reported to be upregulated in human receptive endometrium, which was critical for embryo attachment as an intracellular molecule. Here, we identified Axna2 as a membrane-bound molecule on human endometrial epithelial cells and trophoblast cells, and the outer surface membrane-bound Axna2 was involved in human embryo attachment. In addition, physiological levels of estrogen and progesterone increased the expression of overall Axna2 as well as that in the extracellular surface membrane protein fraction in human endometrial cells. Furthermore, p11 (or S100A10, a member of the S100 EF-hand family protein, molecular weight 11 kDa) was involved in the translocation of Axna2 to the outer surface membrane of endometrial epithelial cells without affecting its overall expression. Finally, the surface relocation of Axna2 was also dependent on cell-cell contact and calcium binding. A better understanding of the function and regulation of Axna2 in human endometrium may help us to identify a potential therapeutic target for subfertile and infertile patients.

Mechanism Matters: A Taxonomy of Cell Penetrating Peptides.

The permeability barrier imposed by cellular membranes limits the access of exogenous compounds to the interior of cells. Researchers and patients alike would benefit from efficient methods for intracellular delivery of a wide range of membrane-impermeant molecules, including biochemically active small molecules, imaging agents, peptides, peptide nucleic acids, proteins, RNA, DNA, and nanoparticles. There has been a sustained effort to exploit cell penetrating peptides (CPPs) for the delivery of such useful cargoes in vitro and in vivo because of their biocompatibility, ease of synthesis, and controllable physical chemistry. Here, we discuss the many mechanisms by which CPPs can function, and describe a taxonomy of mechanisms that could be help organize future efforts in the field.

Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli.

Poly(ethylene terephthalate) (PET) is the most commonly used polyester polymer resin in fabrics and storage materials, and its accumulation in the environment is a global problem. The ability of PET hydrolase from Ideonella sakaiensis 201-F6 (IsPETase) to degrade PET at moderate temperatures has been studied extensively. However, due to its low structural stability and solubility, it is difficult to apply standard laboratory-level IsPETase expression and purification procedures in industry. To overcome this difficulty, the expression of IsPETase can be improved by using a secretion system. This is the first report on the production of an extracellular IsPETase, active against PET film, using Sec-dependent translocation signal peptides from E. coli. In this work, we tested the effects of fusions of the Sec-dependent and SRP-dependent signal peptides from E. coli secretory proteins into IsPETase, and successfully produced the extracellular enzyme using pET22b-SPMalE:IsPETase and pET22b-SPLamB:IsPETase expression systems. We also confirmed that the secreted IsPETase has PET-degradation activity. The work will be used for development of a new E. coli strain capable of degrading and assimilating PET in its culture medium.

Gasdermin D serves as a key executioner of pyroptosis in experimental cerebral ischemia and reperfusion model both in vivo and in vitro.

Even though ischemic stroke is among the leading causes of death worldwide, the pathogenic mechanisms underlying ischemia reperfusion (I/R) brain injury remain unclear. Gasdermin D (GSDMD), as an important factor of pyroptotic death execution downstream of caspase-11 (noncanonical inflammasome) and caspase-1 (canonical inflammasome), may be implicated in I/R injury. The current study aimed to investigate the role and possible underlying mechanisms of GSDMD in pyroptosis during I/R injury. Results indicated that the nucleotide-binding oligomerization domain-like receptors (NLR family) pyrin domain containing 3 (NLRP3) inflammasomes were assembled and activated after middle cerebral artery occlusion/reperfusion (MCAO/R), leading to increased levels of IL-1ß and IL-18. Additionally, GSDMD levels were elevated, and its N-terminal fragment (GSDMD-N) was cleaved to induce pyroptosis after MCAO/R, which was partly dependent on caspase-1 activation and its Asp280 amino acid site. Furthermore, it was found that GSDMD-N could bind to membrane lipids and exhibit membrane-disrupting cytotoxicity, depending on its Glu15 and Leu156 amino acid sites. Nevertheless, the C-terminal fragment of gasdermin (GSDMD-C) exhibited an auto-inhibitory effect on GSDMD-N-induced pyroptosis via binding to GSDMD in the cytoplasm. Taken together, this information suggests that GSDMD may participate in caspase-1-mediated pyroptosis during I/R injury both in vivo and in vitro, which could be a potential therapeutic target to reduce brain I/R injury.

Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin.

The zoonotic pathogen Pasteurella multocida produces a 146-kDa modular toxin (PMT) that enters host cells and manipulates intracellular signaling through action on its Gα protein targets. The N terminus of PMT (PMT-N) mediates cellular uptake through receptor-mediated endocytosis, followed by the delivery of the C-terminal catalytic domain from acidic endosomes into the cytosol. The putative native cargo of PMT consists of a 710-residue polypeptide with three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has an as-yet-undefined function, and C3 catalyzes the deamidation of a specific active-site glutamine residue in Gα protein targets. However, whether the three cargo subdomains are delivered intact or undergo further proteolytic processing during or after translocation from the late endosome is unclear. Here, we demonstrate that PMT-N mediates the delivery of its native C-terminal cargo as a single polypeptide, corresponding to C1-C2-C3, including the MLD, with no evidence of cleavage between subdomains. We show that PMT-N also delivers nonnative green fluorescent protein (GFP) cargo into the cytosol, further supporting that the receptor-binding and translocation functions reside within PMT-N. Our findings further show that PMT-N can deliver C1-C2 alone but that the presence of C1-C2 is important for the cytosolic delivery of the catalytic C3 subdomain by PMT-N. In addition, we further refine the minimum C3 domain required for intracellular activity as comprising residues 1105 to 1278. These findings reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo is delivered intact as C1-C2-C3.

Structural determinants of phorbol ester binding activity of the C1a and C1b domains of protein kinase C theta.

The PKC isozymes represent the most prominent family of signaling proteins mediating response to the ubiquitous second messenger diacylglycerol. Among them, PKCθ is critically involved in T-cell activation. Whereas all the other conventional and novel PKC isoforms have twin C1 domains with potent binding activity for phorbol esters, in PKCθ only the C1b domain possesses potent binding activity, with little or no activity reported for the C1a domain. In order to better understand the structural basis accounting for the very weak ligand binding of the PKCθ C1a domain, we assessed the effect on ligand binding of twelve amino acid residues which differed between the C1a and C1b domains of PKCθ. Mutation of Pro9 of the C1a domain of PKCθ to the corresponding Lys9 found in C1b restored in vitro binding activity for [3H]phorbol 12,13-dibutyrate to 3.6 nM, whereas none of the other residues had substantial effect. Interestingly, the converse mutation in the C1b domain of Lys9 to Pro9 only diminished binding affinity to 11.7 nM, compared to 254 nM in the unmutated C1a. In confocal experiments, deletion of the C1b domain from full length PKCθ diminished, whereas deletion of the C1a domain enhanced 5-fold (at 100 nM PMA) the translocation to the plasma membrane. We conclude that the Pro168 residue in the C1a domain of full length PKCθ plays a critical role in the ligand and membrane binding, while exchanging the residue (Lys240) at the same position in C1b domain of full length PKCθ only modestly reduced the membrane interaction.

Unveiling the Multifaceted Mechanisms of Antibacterial Activity of Buforin II and Frenatin 2.3S Peptides from Skin Micro-Organs of the Orinoco Lime Treefrog (Sphaenorhynchus lacteus).

Amphibian skin is a rich source of natural compounds with diverse antimicrobial and immune defense properties. Our previous studies showed that the frog skin secretions obtained by skin micro-organs from various species of Colombian anurans have antimicrobial activities against bacteria and viruses. We purified for the first time two antimicrobial peptides from the skin micro-organs of the Orinoco lime treefrog (Sphaenorhynchus lacteus) that correspond to Buforin II (BF2) and Frenatin 2.3S (F2.3S). Here, we have synthesized the two peptides and tested them against Gram-negative and Gram-positive bacteria, observing an effective bactericidal activity at micromolar concentrations. Evaluation of BF2 and F2.3S membrane destabilization activity on bacterial cell cultures and synthetic lipid bilayers reveals a distinct membrane interaction mechanism. BF2 agglutinates E. coli cells and synthetic vesicles, whereas F2.3S shows a high depolarization and membrane destabilization activities. Interestingly, we found that F2.3S is able to internalize within bacterial cells and can bind nucleic acids, as previously reported for BF2. Moreover, bacterial exposure to both peptides alters the expression profile of genes related to stress and resistance response. Overall, these results show the multifaceted mechanism of action of both antimicrobial peptides that can provide alternative tools in the fight against bacterial resistance.

The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding.

Colicins are protein toxins made by Escherichia coli to kill related bacteria that compete for scarce resources. All colicins must cross the target cell outer membrane in order to reach their intracellular targets. Normally, the first step in the intoxication process is the tight binding of the colicin to an outer membrane receptor protein via its central receptor-binding domain. It is shown here that for one colicin, E1, that step, although it greatly increases the efficiency of killing, is not absolutely necessary. For colicin E1, the second step, translocation, relies on the outer membrane/transperiplasmic protein TolC. The normal role of TolC in bacteria is as an essential component of a family of tripartite drug and toxin exporters, but for colicin E1, it is essential for its import. Colicin E1 and some N-terminal translocation domain peptides had been shown previously to bind in vitro to TolC and occlude channels made by TolC in planar lipid bilayer membranes. Here, a set of increasingly shorter colicin E1 translocation domain peptides was shown to bind to Escherichia coli in vivo and protect them from subsequent challenge by colicin E1. A segment of only 21 residues, the "TolC box," was thereby defined; that segment is essential for colicin E1 cytotoxicity and for binding of translocation domain peptides to TolC. IMPORTANCE: The Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.

Ligand-induced compaction of the PEX5 receptor-binding cavity impacts protein import efficiency into peroxisomes.

Peroxisomes entirely rely on the import of their proteome across the peroxisomal membrane. Recognition efficiencies of peroxisomal proteins vary by more than 1000-fold, but the molecular rationale behind their subsequent differential import and sorting has remained enigmatic. Using the protein cargo alanine-glyoxylate aminotransferase as a model, an unexpected increase from 34 to 80% in peroxisomal import efficiency of a single-residue mutant has been discovered. By high-resolution structural analysis, we found that it is the recognition receptor PEX5 that adapts its conformation for high-affinity binding rather than the cargo protein signal motif as previously thought. During receptor recognition, the binding cavity of the receptor shrinks to one third of its original volume. This process is impeded in the wild-type protein cargo because of a bulky side chain within the recognition motif, which blocks contraction of the PEX5 binding cavity. Our data provide a new insight into direct protein import efficiency by removal rather than by addition of an apparent specific sequence signature that is generally applicable to peroxisomal matrix proteins and to other receptor recognition processes.