Modes of IgSuperfold Interaction

The archetypal Ig-superfold in immunoglobulins mediates specific recognition through the complementarity-determining region (CDR) loops at one end of the variable domain P-sandwich. However, subsequent IgSF structures and functional studies have highlighted the diversity of potentially functional surfaces on the Ig-superfold. Ig-like domains can interact with a ligand via their P-sheets, via their loop regions, or via a combination of both. A single Ig-like domain may be functional in isolation...

Introduction

Eph receptors represent the largest known family of receptor tyrosine kinases (RTKs) and are activated by interaction with cell-surface ligands, termed ephrins, presented by neighboring cells. The physiological roles of Ephs and ephrins range from early embryonic developmental processes such as cell migration and the establishment of compartment boundaries to processes in the adult brain, such as the regulation of neuronal plasticity. At the cellular level, ephrins guide migrating cells and...

FceRIICD23

No crystal structure is yet available for FceRII, but homol-ogy models have been built for the trimer of C-type lectin domains, based upon crystal structures of highly homologous members of this family 28-30 . The interaction site on IgE Fc is contained within Ce3, but, despite the fact that this domain is glycosylated and binds to the lectin-like domain of FceRII, carbohydrate is not involved 31 . The site in Ce3 is distinct from that of FceRI, and the stoichiom-etry of binding is 2 1 (sFceRII...

ErbB Proteins and Pathological Conditions

Due to their widespread expression and signaling potency, ErbB molecules are involved in a variety of physiological processes (e.g., myelination, implantation, wound healing, mammary development, angiogenesis) and pathological states (airway inflammation, asthma, ulcers, and other gastrointestinal tract diseases). However, the best studied is the oncogenic aspect of the ErbB network in human malignancies. ErbBs were first implicated in cancer upon the characterization of an aberrant form of...

Conclusions and Perspectives

With their bipartite structure already present in primitive marine invertebrates 49 , the GPHr have evolved a specific way to become activated after binding of their hormones to the ectodomain. On the other hand, their membership in the rhodopsin-like family of GPCRs implies that basic molecular mechanisms implicated in the activation of their serpentine domain must be shared with this protein family. We believe that these peculiarities provide a unique opportunity to dissect the molecular...

The Site 3 Interface

The gp130-cytokines possess a third, or site 3, functional epitope that is necessary for receptor activation, but the nature of its interaction with gp130 is unclear 22 . In the structure of the vIL-6-gp130 complex, the site 3 interaction is comprised of an extensive interface between the tips of the vIL-6 four-helix bundle (A B loop and start of the D-helix) and the edge (GF strands) of the upper three-stranded P-sheet of the gp130 IGD (D1) (Fig. 1B). The complementary shape of the interface...

Complex Formation with TGFP Is Different than for BMP2

Our understanding of the receptor-ligand complex, with well-separated type I and type II binding epitopes and assumed to be valid for the entire TGFP superfamily, had to be revised with the publication of the crystal structure of TGFP3 in complex with TGFpR-IIec 19 . Surprisingly, the receptor does not bind to a region corresponding to the knuckle epitope on BMP-2. Instead, it interacts with a hydrophobic cleft in the fingertips of the ligand and is in close proximity to the expected type I...

Avoiding the Menace of Toxins in the Real World Outside the Laboratory

Our enthusiasm for using the toxins in biomedical research was tempered by news of a most tragic case of microcystin poisoning in 1996. More than 100 dialysis patients in Caruara, Brazil, were infused with microcystic water and most died of liver failure 26 . The scale of tumor promotion and liver damage worldwide is more difficult to assess. However, microcystin levels above the WHO limit (1 g liter) and suspected human and animal poisonings are often reported 8 . How can researchers who...

Other PTP Inhibitors

PTP1B was the founding member of the PTP family, and a large amount of structural and mechanistic information is Figure 3 Structures of potent and selective bidentate PTP1B inhibitors. Figure 3 Structures of potent and selective bidentate PTP1B inhibitors. available for PTP1B. Furthermore, biochemical and genetic data suggest that PTP1B is a negative regulator for both insulin and leptin signaling. Consequently, most of the PTP inhibitors that are reported in the literature are directed to...

Eph Receptor Signaling Via Rho Family GTPases

The small GTP-binding proteins of the Rho GTPase family, including RhoA, Rac, and Cdc42, are important regulators of the actin cytoskeleton and neuronal morphogenesis 9 . Rho GTPases are molecular switches that cycle between an active GTP-bound state and an inactive GDP-bound state. GTP-bound forms are converted into GDP-bound forms by the action of GTPase-activating proteins (GAPs), whereas guanine nucleotide exchange factors (GEFs) perform the opposite conversion. The Rho-specific GAP,...

Physical Models of Activation Turning a Screw through a Bolt

What kind of physical structure can account for S4 packing and motion Similar to the short (12 A) and narrow ( 5 A) ion-selectivity filter that opens to water on either end (see Chapters 34 and 36), the short (13.5 A) gating canal must also have water-filled crevices at its ends. Unlike the selectivity filter, which is designed for a high rate of flux ( 107 per second) and so employs loose coordination of permeant ions by carbonyl oxygens, the gating canal probably employs strong electrostatic...

References

J. (1979). Three-dimensional structure of immunoglobulins. Annu. Rev. Biochem. 48, 961-997. 2. Amit, A. G., Mariuzza, R. A., Phillips, S. E., and Poljak, R. J. (1986). The three-dimensional structure of an antigen-antibody complex at 2.8 A resolution. Science 233, 747-753. 3. Sheriff, S., Silverton, E. W., Padlan, E. A., Cohen, G. H., Smith-Gill, 5. J., Finzel, B. C., and Davies, D. R. (1987). Three-dimensional structure of an antibody-antigen complex. Proc. Natl....

The Cyclic Nucleotide Gated Channels

The CNG channels of both photoreceptors and olfactory neurons are nonselective cation channels that conduct Na+, K+, and Ca2+ when activated (opened) by cyclic nucleotide binding. Their single-channel conductance is typically greater than 20 pS when divalent cations are absent, indicating a relatively high rate of Na+ and K+ flux. Under physiological ionic conditions, the channels pass an inward current that depolarizes the membrane, but external Mg2+ potently blocks current flow through the...

IL4 Hormone Induced Receptor Activation

IL-4 is a pleiotropic hormone that mediates several important regulatory responses in the immune system (9A, 10A). The hormone belongs to the short-chain, four-a-helical bundle class of cytokines (4), which is a distinctly different class than the long-chain cytokines represented by GH and PRL. The IL-4 signaling cascade is triggered by a sequential hormone-induced aggregation of two distinct receptor chains (12A, 13A). The so-called a-chain receptor binds first to IL-4 with high affinity (150...

IRSProtein Signaling in Growth Nutrition and Longevity

The disruption of IRS-proteins in mice and flies reveals their role of coordinating multiple biological processes, including growth, nutrition, and fertility. The framework relating IRS-proteins to these biological processes might be easier to establish in Drosophila, because fewer signaling protein are involved. Deletion of Chico, the Drosophila IRS-protein, causes female sterility as well as reduced somatic growth and increased lipid storage 55 . Moreover, specific mutations of the binding...

Summary

Protein phosphorylation frequently induces specific protein-protein interactions mediated by specific phospho-tyrosine- or phosphoserine threonine-binding domains. These phospho-dependent interaction domains are important for the specificity of signal transduction downstream of cell-surface receptors and serve as the prototype for a large family of binding modules that control many aspects of cellular organization. Dr. Piers Nash is a senior research fellow of the Canadian Institutes of Health...

PTyr Surrogates as PTP Inhibitors

X-ray crystallographic studies have revealed a very similar active site (the pTyr binding site) for PTPs. In the substrate-bound form of PTP1B, the terminal non-bridge phosphate oxygens of pTyr form an extensive array of hydrogen bonds with the main-chain nitrogens of the PTP signature motif (residues 215-221) and the guanidinium side chain of Arg221. The phenyl ring of the pTyr is engaged in hydrophobic interactions with the active-site cavity, formed by the nonpolar side chains of Val49,...

Conclusion

The biology of signaling features a whole range of interactions, from weak to strong. Dissociation constants are found in the nM to mM range 10 . Both the complementarity of the physicochemical and geometrical properties of the interface and the flexibility of the surfaces of the receptor and signalling molecule contribute to the binding free energy. Structural data and currently available computational methods appear inadequate to estimate the binding energy or specificity of an interaction....

Structural Mechanistic Studies

Flawe Antena

Structural studies of the insulin receptor have been limited largely to the cytoplasmic portion of the P-subunit. Within the cytoplasmic portion are 36 residues in the jux-tamembrane region (membrane-proximal), 295 residues in the tyrosine kinase domain, and 72 residues in the C-terminal tail (Fig. 1). Crystal structures of the insulin receptor kinase domain (IRK) have been determined in different states of phosphorylation 8,9 and for several mutants 10 (S. R. Hubbard, unpublished data). IRK...

Biological Roles of Carbohydrate Recognition

Carbohydrates, in the form of oligosaccharides or glyco-conjugates, are found on the cell surfaces and extracellular proteins of virtually all living organisms. Although roles for carbohydrates in endogenous physiological interactions had long been suspected, it was not until the 1970s, with the discovery of the hepatic asialoglycoprotein receptor and Man-6-phosphate-mediated intracellular protein targeting, that firm evidence for such roles began to emerge. Since then, a number of animal...

James A Wells

Cells within multicellular organisms communicate via extracellular mediators either through diffusible molecules or by direct cell-cell contact. These extracellular signals are interpreted for the most part by specific membrane receptors that in turn trigger intracellular machinery that directs the cellular response. The past decade has seen an explosion in our understanding of the molecular basis for membrane receptor signaling. Part I in this Handbook surveys the diversity of mechanisms for...

Conclusions

The ability of surface membrane receptors to relay extrinsic signals to the cell is often dependent upon their localization to functionally relevant sites on the cell surface. In activation pathways that are regulated by the convergence of positive and negative signals, differential localization of stimulatory and inhibitory receptors can be critical for regulating a balance between activation and inhibition. The localization patterns of CD28 and CTLA-4 exemplify this concept. Here, the...

ATM and ATR Signalers of Genome Damage

A homozygous deficiency of ATM in humans leads to ataxia-telangiectasia (A-T), a debilitating disorder in which progressive loss of motor coordination (ataxia) is brought about by the gradual loss of Purkinje cells in the cerebellum 33 . In addition, A-T patients have an increased cancer incidence, and cells derived from these individuals are hypersensitive to ionizing radiation and to chemical agents that induce DNA double-strand breaks 33 . Notably, whereas normal cells delay progression...

Control of the CFTR Chloride Channel by PP2C

Cystic fibrosis is the most common autosomal lethal genetic disease among Caucasians, and the protein product of the disease-causing gene is known as the cystic fibrosis transmembrane conductance regulator (CFTR) 15 . CFTR is the known only member of the ATP-binding cassette (ABC) transporter family that forms an ion channel. It is located mostly in the apical membrane of epithelia, where it mediates transepithelial salt and liquid movement. CFTR consists of five domains two transmembrane...

Serthr Kinase Inhibitors

Among the few hundred Ser Thr kinases a handful are involved in transmitting the signals of upstream PTKs, and their activity is essential for cell proliferation and the onset of anti-apoptotic signaling (Fig. 7). Their abnormal enhanced activities are augmented by the deletion of negative regulators such as protein inhibitors of Cdks and the deletion of the tumor suppressor PTEN, the negative regulator of the PI3' kinase pathway. Thus, the activities of these kinases are enhanced by the...

Molecular Pathophysiology

Amino acid substitutions at each of 20 separate positions cause constitutive activation of TSH or LH CG receptors 9 . The TSH receptor has been particularly fertile in activating mutations because of the fact that somatic mutations leading to activation of the TSHr cause a readily detectable thyroid phenotype (i.e., autonomous toxic adenomas) 10 . Much less frequent are germline mutations with similar activating effects. When affecting the TSHr or LH CGr, they cause autosomal dominant...

Novel Features in the Structure of Apo2LTRAIL

Two independent crystal structures of unbound Apo2L TRAIL show that it resembles the TNF trimer in its basic architecture 5,6 . Apo2L TRAIL has two unusual features with respect to other TNF family members. First, it has a distinct, long loop between strands A and A' (residues 130 to 150) that traverses its surface but is poorly ordered or even disordered between residues 131 and 143 (Fig. 1). Second, a novel zinc-binding site buried in the trimer interface is formed by the single cysteine...

Binding of the yChain Receptor

The y-chain exhibits no measurable binding to either IL-4 or the a-chain receptor alone (8A) and, as is observed for hGH-hGH-R homodimerization, the binding of the second receptor is facilitated by structural properties generated by formation of the 1 1 intermediate. A major difference, however, is that, whereas the second hGH receptor binds to the intermediate 1 1 complex with high affinity (4 nM) (Bernat and Kossiakoff, submitted), the y-chain binds to the IL-4-a-chain receptor 1 1 complex...

Lipid Signaling

The elucidation of cell signaling mechanisms and the variety of molecules that are employed in these myriad of processes is particularly well exemplified by the lipid messengers. Except for the above mentioned steroid hormones, lipids have long been thought to function mainly in energy metabolism and membrane structure. This last decade has culminated in the broad recognition that membrane phos-pholipids provide many of the important cell signaling molecules via phospholipases and lipid...

Receptor Receptor Interactions

A conserved structural element of the ligand-induced homodimerization of prolactin and growth hormone receptors is a set of extensive contacts between their C-terminal domains. This receptor-receptor interface was described in detail for the hGH hGH-R 1 hGH-R2 ternary complex 3,8 and modeled for the hGH-hPRL-R ternary complex 3 . Although the topology of the C-terminal domains of the rPRL-R is virtually identical to that of the C-terminal domain of hGH-R, the receptor-receptor interfaces in...

Carbohydrate Structure and Diversity

The structural diversity characteristic of the oligosaccha-rides found in nature stem principally from three sources (1) a large number of monosaccharide types, (2) the multiple ways in which the monosaccharides can be linked Figure 1 Structural representation of the sulfated sialyl Lewis x tetrasac-charide, NeuAc, Gal, GlcNAc, and Fuc label the monosaccharide moieties N-acetylneuraminic acid, galactose, N-acetylglucosamine, and fucose, respectively. Figure 1 Structural representation of the...

Flu Virus Role of NA

Influenza remains a major cause of mortality and morbidity worldwide. Vaccination affords some protection but must be reformulated each year based on a prediction of the most likely strains circulating in the coming flu season. The antigenic drift and shift characteristic of the virus limits the effectiveness of the vaccine, and some warn of a re-emergence of a catastrophic pandemic strain such as occurred in 1918 the so called Spanish flu 1 . Two antiviral drugs (amantadine and rimantadine)...

Functional Aspects of PTPs in Health and Disease Bioinformatics

Phosphorylation of cellular proteins regulates most signal transduction processes, and many diseases have been associated with abnormal phosphorylation patterns. An intricate balance and regulatory machinery are required to maintain the exact levels of phosphorylation. How can less than 50 different classic PTPs be sufficient to regulate and fine-tune phosphotyrosine levels in a human from conception, during embryonic life, and through childhood, puberty, and adulthood In addition to inherent...

PTPs and Human Disease

The importance of PTPs to the control of signal trans-duction has been further reinforced by numerous examples in which the disruption of normal PTP expression or function has been implicated in human disease. A summary of developments in this area is presented in Table 2. In light of the large number of PTKs that have been shown to play a role in oncogenesis, it was anticipated that many of the PTPs may be the products of tumor suppressor genes. Although PTPs have been linked to inhibition of...

Binding Determinants of the IR

Insulin is thought to have two distinct receptor binding surfaces, with one site encompassing at least the residues G1, E4, Q5, and N21 of the insulin A-chain and the other site being made up of residues V12, Y16, F24, F25, and Y26 of the B-chain other residues apart from those listed above are almost certainly involved in this interaction 6 . Binding of insulin to the native, dimeric IR is characterized by curvilinear Scatchard plots and the phenomenon of negative cooperativity, both...

Biological Functions of Shps

Genetic models for murine Shp1 and for Shp2 orthologs in mouse, Drosophila, and C. elegans have been invaluable for defining the biological functions of the Shps. The phe-notypes of Shp-deficient organisms will be described briefly here more complete descriptions are available in several other reviews. Two naturally occurring point mutations exist in the murine Shp1 gene, each of which causes abnormal splicing of Shp1 transcripts 91,92 . The motheaten (me) allele generates an early frameshift...

Biological Implications of Transient Receptor Dimerization

Functional and structural information suggests that the role of receptor homodimerization is more complicated than simply bringing the cytoplasmic elements of the receptors together. For instance, structural studies of erythropoietin (EPO) and its receptor (EPO-R) indicate that a function of the hormone is to establish a fairly exact receptor alignment, as well as to induce dimerization 33-36 . Based on patterns of cross-hormone and cross-species activities and the known structural differences...

Concluding Remarks

In this brief overview, we have summarized much of the salient observations that support the view that oligomeriza-tion is a prevalent and perhaps universal requirement for transmembrane receptor function. There is clearly growing evidence, in part attributable to better technology, that many of these oligomeric structures form independent of ligand and that activation by ligand binding is not due to association of the receptor protomers but rather allows them to seek a different juxtaposition...

Darchone Chow Lena Brevnova Xiaolin He and K Christopher Garcia

Department of Microbiology and Immunology and Department of Structural Biology, Stanford University School of Medicine, Stanford, California The gp130-cytokine system has been the subject of extensive protein structure-function studies aimed at elucidating the basis of ligand recognition and receptor activation. A longstanding question has been the architecture of the higher order signaling assembly. It is clear from functional studies that the paradigm of gp130-cytokine recognition will differ...

Evolution of a Phospho Dependent Docking Protein

As animals have become more complex, modular signaling proteins appear to have evolved through the acquisition of additional modules or binding sites. For example, Shc is a docking protein with a C-terminal SH2 domain and an N-terminal PTB domain flanking a central region containing a proline-rich section and an adaptin binding motif. The distinct binding properties of SH2 and PTB domains allow Shc to interact with multiple phosphotyrosine-containing motifs on cell-surface receptors. The...

Functional Consequences of GPCR Endocytosis

Role in Rapid Desensitization of GPCRs In many cases, endocytosis is not thought to play a primary role in mediating rapid desensitization of many GPCRs, although the precise role of endocytosis in this process may depend on receptor expression level. Endocytosis of -opioid receptors does not contribute significantly to functional desen-sitization in cells expressing relatively high levels of receptor protein but does appear to cause desensitization in cells expressing lower levels of receptor...

General Processes of GPCR Regulation

Desensitization and Resensitization Rapid Regulation of the Functional Activity of Receptors Many GPCRs are regulated very rapidly after agonist-induced activation, a process that has been characterized in considerable detail in studies of the P2 adrenergic receptor (P2-AR) 3-5 . Upon binding of agonist the P2-AR promotes guanine nucleotide exchange on its cognate heterotrimeric G protein (Gs), which thereby activates downstream effectors such as adenylyl cyclase. Receptor-mediated signaling...

Hormone Specificity and Cross Reactivity Determine Physiological Roles

Binding to the two structurally distinct sites on the hormone, while using the same binding determinants, requires the receptor binding surfaces to undergo significant local conformational change 8,18 . The structural requirement is further expanded by specificity factors 9 . The biology of pRL and GH is integrated on many levels 20 however, over the 400 million years since pRL and GH diverged from a common gene parent, evolution has built in different regulating components distinguishing them...

IgEReceptor Interactions

The crystal structure of the complex between the two extracellular domains of FceRI a-chain and a subfragment of IgE Fc consisting of the Ce3 and Ce4 domains 21 (termed here Fce3-4 to distinguish it from the whole Fc that includes the Ce2 domains) showed essentially the same topology of interaction (Fig. 2C) as seen for the IgG Fc FcyRIII complex. The Ce3 domains of Fc open up upon binding to the receptor, compared to the uncomplexed state 22,23 , but there is little change in the angle between...

Implications of the vIL6gp130 Tetramer Structure for the Active Gcsfgcsfr Extracellular Signaling Complex

A crystal structure has been reported for GCSF in complex with the CHR (domains 2 and 3 also called the BN and BC domains, respectively) of the GCSFR (Fig. 2A) 2 . The structure revealed both expected and unexpected results. The complex is a 2 2 tetramer, with each GCSF molecule interacting with each receptor through the canonical site 2 epitope (also called major interface) on the face of the A and C helices (Fig. 2A) 2 . However, the unexpected result was that the two GCSF GCSFR complexes...

Interleukin4 and IRS2 Signaling

IRS2 was originally identified as a tyrosyl-phosphorylated protein called 4PS in interleukin-4 (IL-4)-stimulated cells 58 . 4PS was also stimulated by insulin and insulin-like growth factor 1 (IGF1), and IL-4- or insulin-insensitive 32D cells lacking 4PS were rescued by expression of IRS1 59 . Although 4PS was found to comigrate with IRS1 during sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), IRS1 antibodies failed to immunoprecipitate it, suggesting that 4PS was a...

Jee Y Chung Young Chul Park Hong Ye and Hao Wu

Department of Biochemistry, Weill Medical College of Cornell University, New York, New York Unlike receptors with intrinsic kinase activity in their intracellular domains or in direct association with intracellular enzymes, members of the tumor necrosis factor (TNF) receptor superfamily and the interleukin-1 (IL-1) receptor Toll-like receptor (IL-1R TLR) superfamily use adapter proteins to couple to enzymatic activation and signal amplification. The TNF-receptor-associated factors (TRAFs),...

Ligand Receptor Complexes

Tumor necrosis factor a binds tightly to ECD domains of TNF-R1 and TNF-R2 with dissociation constants in the range of 0.1 to 1 nM 22,32 . The structure of the TNF-P-TNF-R1 complex, which contains three receptor ECDs and one TNF-P trimer, immediately suggests a mechanism for the proximal events of signal transduction 28 (Fig. 3). Within the complex, each ECD binds to the crevice formed at the interface between two neighboring, triad-related TNF-P subunits. The receptors do not contact each...

Medical Implications of the AMPK System

Type 2 diabetes, which affects over 100 million people worldwide, is a hyperglycemic condition caused by reduced glucose uptake by muscle and increased glucose production by liver. Physical exercise is known to provide protection against its development, and because AMPK is activated by exercise it regulates the activity and expression of the insulin-sensitive glucose transporter GLUT4 and inhibits expression of enzymes of gluconeogenesis 30 , this suggesting that AMPK could be a promising...

Multiple Virus Encoded 7TM Receptors

More than 20 G-protein-coupled receptors have been identified in various herpes- and poxviruses (Table 1 and Fig. lA). Most of these receptors display key structural elements, that identify them as belonging to the family of chemokine receptors. In general, however, it is impossible to identify a specific endogeneous chemokine receptor as the original scaffold hijacked by the virus. The extensive subsequent mutational effort performed by the virus which has generated the desired pharmacological...

Russsell F Doolittle

Center for Molecular Genetics, University of California, San Diego, La Jolla, California Fibrinogen is an extracellular protein found in significant concentrations in the blood plasmas of all vertebrate animals. It is a large, multi-domained protein, some portions of which share common ancestry with lectins and other cyto-tactic proteins found throughout the animal kingdom 1 . Although the principal role of fibrinogen has to do with its polymerization into fibrin clots, the protein also...

Structural Basis for Receptor Homodimerization

Tertiary structure plays a role in how the hormone regulates receptor activation. The hormones in this family are long chain four a-helix bundle proteins 4,17 . A notable feature of their tertiary structure is that it contains no symmetry that might support equivalent binding environments for the receptors. How the two receptors bind to the asymmetric hormone was first revealed from the crystal structure of human growth hormone bound to the extracellular domain (ECD) of its receptor (hGH-R) 8 ....

Signaling at Periplasmic Ligand Binding Domain

In contrast to many other receptors, such as growth hormone receptors, bacterial chemotaxis receptors do not signal by horizontal aggregation of the receptor monomers instead, ligand binding induces small conformational changes, which are assumed to be transmitted through the transmembrane helices to the cytoplasmic domain and to affect the phospho-rylation rate of the bound histidine kinase (recently reviewed by Falke and Hazelbauer 29 ). Crystal structures of the ligand binding domain of a...

The Growth Hormone Family of Hormones and Receptors

Growth hormone (GH), placental lactogen (PL), and prolactin (PRL) regulate an extensive variety of important physiological functions. While GH biology generally centers around the regulation and differentiation of muscle, cartilage, and bone cells, it is the PRL hormones and receptors that display a much broader spectrum of activities, ranging from their well-known effects in mammalian reproductive biology to osmoregulation in fishes and nesting behavior in birds 1 . Within the cytokine...

The Immunoglobulin Superfamily

The P-sandwich topology, first identified in the domains of immunoglobulin (Ig) structures and hence termed the Ig-fold, is one of the most common structural motifs in the proteins of multicellular organisms. The immunoglobulin superfamily (IgSF) is comprised of proteins that contain at least one Ig domain. This motif characteristically occurs within the extracellular portions of proteins and frequently mediates recognition events. Indeed, the evolution of IgSF proteins appears to be linked to...

The Redundant Chemokine System Is an Optimal Target for Viral Exploitation

Chemokines are chemotactic cytokines, which primarily control the migration but also the activation and differentiation of all subsets of leukocytes and play important roles in angio-genesis, organogenesis, and carcinogenesis 1 . Chemokines act through a large family of G-protein-coupled receptors, which are divided into subfamilies of CXC, CC, and CX3C receptors. This nomenclature refers to a fingerprint sequence in the ligands where the first two Cys residues are either neighbors (CC) or...

The Regulation of GSK3 Activity by Insulin and Growth Factors

GSK3 can be inhibited via the phosphorylation of a serine residue near the N terminus of the protein (Ser21 in GSK3a, Ser9 in GSK3P) 22 . This serine lies in an Arg-Xaa-Arg-Xaa-Xaa-Ser sequence, which is a consensus motif for phosphorylation by several protein kinases that are components of different signal transduction pathways (Fig. 1). Protein kinase B (PKB, also called Akt) inhibits GSK3 in response to signals that activate class I phos-phatidylinositol (PtdIns) 3-kinases and elevate the...

Thermodynamic Mapping of Antigen Antibody Interfaces

In contrast to the wealth of structural information on antigen-antibody and other protein-protein interfaces, the available data on the thermodynamics of the association reactions are far more limited. Indeed, our current view of the energetics of protein-protein association is largely based on detailed mutagenesis and binding studies of only a few complexes 15 . We have studied the binding of monoclonal antibody D1.3 to two structurally distinct ligands its cognate antigen, hen egg white...

Ga Subunits

Adenylyl Cyclase Mg2

Ga subunits are members of the Ras superfamily, which also includes translation elongation factors and the components of the signal recognition apparatus. In mammals, the family of Ga isoforms is encoded by 16 genes these can be sorted into four closely related homology groups or classes named for representative members of each class Gas, Ga , Gaq, and Ga12 (Fig. 1). Two variants of Gas are generated by alternative mRNA splicing. Each member of the Ga family interacts specifically with one...

Insulin Receptor Domain Structure

The insulin receptor (IR) is a glycosylated, disulfide-linked homodimer, with each monomer being made up of an a-chain that is entirely extracellular and a P-chain that spans the cell membrane once. The a-chain contains the insulin-binding determinants of the receptor, while the intracellular portion of the P-chain includes a protein-tyrosine kinase domain and domains involved in binding signal transduction proteins. The aP monomer of the IR is encoded by a gene with 22 exons alternative...

Virus Encoded Proteins Are Developed through Targeted Evolution In Vivo

Large DNA viruses, in particular herpes- and poxviruses, have evolved a number of proteins that function as mimics of or as decoys for endogenous proteins of the host organism. Often the virus uses such proteins to evade key components of the immune system. The virus-encoded proteins are elegant examples of targeted evolution, where the virus has captured a gene from its host and through combinatorial chemistry varied its structure and thereby its function randomly through mutagenesis. Unlike...

Insulin Signaling to Glucose Transport

A major, if not the major, endpoint of insulin signaling is the stimulation of glucose uptake (transport) in muscle and fat, the focus of the remainder of this chapter. Currently, at least two distinct pathways (Fig. 3) have been implicated in this process. Both pathways may be required for translocation of the insulin-regulated glucose transporter, GLUT4, a 12-transmembrane-spanning protein, from a vesicular storage compartment (GSV) within the cell, to the plasma membrane. Its insertion makes...

IRSProteins and Insulin Signaling

Insulin and IGF1 receptors, like the receptors for other growth factors and cytokines, are composed of an extracellular ligand-binding domain that controls the conformation and activity of the intracellular tyrosine kinase 9,10 . Unlike most receptor tyrosine kinases that are activated upon ligand-induced dimerization, insulin and IGF1 receptors exist as inactive covalent dimers composed of two extracellular a-subunits and two transmembrane P-subunits. Insulin and IGF1 bind between the two...

GSK3 and Cancer

Many of the components of the Wnt signaling pathway are over-expressed or mutated in different tumors 38 . For example, virtually all colon tumors arise from an initiating mutation in the APC gene (85 ) or in the P-catenin gene (10-15 ) that makes P-catenin resistant to degradation. Axin mutations occur in hepatocellular carcinomas and FRAT1 in T-cell lymphomas. Alterations in these components would be predicted to lead to inappropriate accumulation of P-catenin. These observations have...

Regulation by D2 Domain

The membrane-distal phosphatase domain of RPTPa (D2) is highly conserved among all RPTPs and exhibits little or no phosphatase activity, despite the fact that most D2 domains possess the catalytic cysteine residue required for pTyr turnover. The X-ray crystal structures of both D1 and D2 domains of LAR 8 and RPTPa 1 (Sonnenburg et al., in preparation) reveal that both phosphatase domains share a very similar overall three-dimensional architecture. The lack of D2 enzymatic activity appears to be...

Src PTKs

Two members of the Src PTKs, Lck and Fyn, have been implicated in T-cell antigen receptor function and development. Studies of lymphocytes lacking the Lck and Fyn members of the Src family of PTKs have implicated Lck and, to a lesser degree, Fyn, as being responsible for the phosphorylation of the T-cell receptor (TCR) ITAM sequences 6,10-13 . Fyn was the first cytoplasmic PTK Handbook of Cell Signaling, Volume 1 Figure 1 Schematic representation of PTKs in T-cell antigen receptor function. 1...

Structural Features of G Protein Activation

The rate-limiting step in G protein activation is the release of GDP from the nucleotide-binding pocket. GDP is spontaneously released from the heterotrimeric G protein at a rate that varies depending on the type of Ga subunit. However, in the basal state, the rate of GDP release is much lower than the rate of GTP hydrolysis, and GPy binding controls this state. GDP release is greatly facilitated by receptor activation of the G protein 11 . Mutations in the TCAT sequence of Ga in the P6-a5 loop...

Introduction to Bioinformatics

Removal of phosphate from phosphotyrosine residue (pTyr) on cellular proteins plays a key role in many different signaling pathways and is catalyzed by three classes of enzymes 1-6 (1) classic protein tyrosine phosphatases (PTPs), (2) dual-specificity phosphatases (dsPTPs), and (3) low-molecular-weight phosphatases (LMW-PTPs). The classic (tyrosine-specific) PTPs, which are the focus of this bioinformatics analysis, have traditionally been classified into receptor-like and intracellular PTPs...

Structure of CaMKII

CaMKII was first purified from rat brain homogenates 6,7 and shown by study of its hydrodynamic properties to be a dodecameric hetero-oligomer of two subunits, termed alpha (50 kDa) and beta (60 kDa) 6 . These subunits are highly homologous to each other and are both catalytic. They appear to assemble together into dodecamers that contain the same average proportions of a- and P-subunits as are present at the time of synthesis. Thus, the holoenzyme composition in a given cell is not homogenous...

TCRpMHC Interaction

Whereas in humoral immunity antibodies identify anti-genic molecules as distinct entities, in the cellular response TCRs recognize antigenic peptide fragments only when presented by an appropriate MHC molecule. A fundamental Handbook of Cell Signaling, Volume l Figure 1 Schematic representation of the components in a class I TCR pMHC CD8 CD3 signaling complex. The heavy chain consists of the a a3 domains, to which the light chain P2-microglobulin (P2m) is noncovalently attached. The peptide-MHC...

Protein Phosphatase 1 PP1

Protein phosphatase 1 belongs to the PPP family of phosphatases and is involved in the regulation of a wide variety of cellular processes ranging from intermediary metabolism to apoptosis. In mammals, three genes code for four or five highly conserved ( 90 ) isoforms of catalytic subunits of PP1 (PP1c), PP1ca1 and a2, PP1c5 (also called PPlc ) and PP1cy1 and y2, the subscripts indicating forms generated by alternative splicing. In the cell, PP1c does not exist as a free monomer but is present...

The Future Ion Channels as Electrosomes

Beyond the classical pore-forming subunits and auxillary subunits that comprise ion channels, it is becoming ever more clear that, in real biological settings, ion channels are part of large protein networks. These networks include cytoskeletal components, signaling proteins such as protein kinases and phosphatases, and channel-associated proteins that recruit these signaling molecules to the channel to modify its function. To understand the biological structure of ion channels, it will be...

General Principles of Control

Allosteric regulation is a classic widespread mechanism of control of protein function effectors bind to regulatory sites distinct from the active site, inducing conformational changes that profoundly influence the activity 7 . Allosteric effectors typically bear no structural resemblance to the substrate of their target protein. This form of regulation explains how end products of metabolic pathways could act at early steps of the pathway to exert feedback control. In protein kinases,...

Protein Serine Threonine Phosphatases of the PPM Family

Protein phosphatases of the PPM family are present in both eukaryotes and prokaryotes for which the defining member is PP2C. Biochemically, the PPM family was distinguished from the PPP family by its requirement for divalent metal ions (Mg2+) for catalytic activity, although it is now known from crystal structures that both PPP and PPM phosphatases catalyze dephosphorylation reactions by means of a binuclear divalent metal center. Within the PPM family, the PP2C domain occurs in numerous...

Integrins Nucleate the Formation of Multi Protein Complexes

A central function of the integrins is to mediate a structural linkage between the dynamic intracellular cytoskeleton and the ECM. More that 50 proteins have been identified either as direct integrin-binding proteins or as proteins that localize to adhesion complexes (e.g., focal adhesions) 4 . The proteins found in adhesion complexes fall into two broad categories those that serve a structural role to anchor and regulate the actin cytoskeleton and those that are responsible for...

Eukaryotic Ion Channels at High Resolution Divide and Conquer

Bacterial channels have provided insight into the guts of ion channel permeation machineries, revealing the intimate details of permeation pathways that are likely to be conserved and recapitulated in their larger eukaryotic cousins. In contrast to prokaryotic membrane proteins (which are difficult to obtain in their own right), eukaryotic membrane proteins are currently extremely difficult to obtain in the quantities required for high-resolution study. Eukaryotic channels often contain a host...

Protein Lipidation

Protein N-myristoylation is the addition of myristic acid to a protein through an amide linkage to an N-terminal glycine residue. Modification of the protein occurs cotranslationally upon removal of the initiator methionine by methionylpepti-dase 5 , or posttranslationally following proteolytic cleavage that exposes an N-terminal glycine 6 . N-myristoylation is a stable modification. Examples of signaling proteins that are N-myristoylated can be found in Table 1. Prenylated proteins are...

Site1 and Site2 Are Structurally and Functionally Coupled

It is likely that this new binding solution for hGHv-hGH-R2 is triggered by a structural mechanism linking site 2 to a subset of the mutations in site 1 introduced in the phage display experiments. It is noteworthy that the structurally distinct conformation of hGHv at site 2 was under no selection pressure and supports binding of the second receptor as tightly as in the wt complex. A specific example of the structural coupling is observed from the altered roles of Asp116 in site 2 of the...

Constitutive Signaling through Altered Pathways

The virus-encoded receptors have often chosen G proteins and downstream effector molecules different from those of the endogenous chemokine receptors. Moreover, whereas most endogenous chemokine receptors are rather silent in the absence of agonist, the virus-encoded receptors often display clear constitutive signaling activity, which may or may not be subject to further fine-tuning or regulation by endogenous ligands. ORF74, also named KSHV-GPCR (i.e., Kaposi's sarcoma-associated herpesvirus...

The Substrate Specificity of GSK3

Soon after its discovery, it was noted that GSK3 could only phosphorylate glycogen synthase efficiently if glycogen synthase had already been phosphorylated by CK2 6 . Phosphorylation by CK2 did not inhibit glycogen synthase, but primed this enzyme for phosphorylation by GSK3. Elegant studies by Roach and his colleagues then established that the substrate specificity requirements of GSK3 are unique the protein kinase phosphorylating serine and threo-nine residues that lie in Ser...

The Consensus Binding Site on Fc

Superposition of the binding site footprints of the four natural IgG-Fc binding domains reveals the presence of a common surface patch on the Fc surface (Fig. 3a). Just six side chains are involved in forming this saddle-shaped consensus site between the 250's loop of the CH2 domain and the 430's loop of the CH3 domain. Together these side chains Figure 3 Superposition of the binding site footprints on IgG-Fc for the natural Fc binding proteins (a). Surface atoms are colored red, yellow, light...

Budding Yeast Cdc14 is Essential for Exit from Mitosis

Following their association with B-type cyclins, the activation of cyclin-dependent kinases (Cdk) triggers the onset of mitosis. At anaphase after sister chromatids have separated, mitotic Cdks must be inactivated in order for cells to exit from mitosis. During exit from mitosis, cells restore the nucleus to its premitotic state (e.g., disassemble the mitotic spindle) and prepare for cytokinesis (for review, see Morgan 5 ). A prevailing mechanism for mitotic Cdk inactivation is the regulated...

Initiation of BCR Signaling Is Controlled by Redox Regulation

How signals are initiated upon antigen binding to the BCR is not completely understood and is still a matter of controversy. It has been found that the three protein tyrosine kinases (PTKs) Syk, Lyn, and Btk, as well as the adaptor protein SLP-65 BLNK and PLC-y2, are required for an optimal calcium response following engagement of the BCR (Fig. 1A) 6 . In the absence of Syk and Btk, no calcium response is observed, but in the absence of the Src-family kinase Lyn the amplitude of the calcium...

The Structure of the B Cell Antigen Receptor

The BCR is composed of two protein modules, the membrane-bound immunoglobulin (mIg) molecule and the Ig-a Ig-P heterodimer, which mediate antigen binding and signaling, respectively (Fig. 1A) 2,3 . The proper assembly between the mIg molecule and the Ig-a Ig-P heterodimer in the membrane of the endoplasmic reticulum is a prerequisite for the transport of the BCR to the cell surface. All five major classes of mIg (mIgM, mIgD, mIgG, mIgA, and mIgE) are associated with the Ig-a Ig-P heterodimer,...

Catalytic Activities of the PPP Family Members

PPPs dephosphorylate phosphoserine and phosphothreo-nine residues in proteins in vivo and in vitro. Mammalian PP1, PP2A, and PP2C have also been shown to dephosphorylate histidine residues in proteins in vitro 14 . Mammalian PPPs expressed in Escherichia coli display properties that are slightly different from the native enzymes, including dependence or partial dependence on divalent metal ions such as Mn2+ or Mg2+ 15 . Bacterially expressed mammalian Ppplc also exhibits protein-phosphotyrosine...

Interaction Domains A Common Theme in Signaling

Interaction domains are essential in signaling from many different types of cell-surface receptors, as well as in cellular events such as the cell cycle, protein and vesicle trafficking, targeted protein degradation, DNA repair, and control of the cytoskeleton. Thus, the SH2 domain serves as a prototype for a growing family of protein-interaction modules (Table 1), some of which specifically recognize posttranslationally modified motifs, in a fashion akin to the selective binding of SH2 and PTB...

Chemokine Structure and Function

Although their discovery has been relatively recent (mostly within the past 15 years), chemokines and their receptors have rapidly become appreciated for their impact on health and disease. They are involved in a broad variety of natural biological processes, including development, inflammation, immunity, and angiogenesis. In addition, these sequences have been corrupted by pathogens to subvert the innate and adaptive immune responses. This review provides a brief introduction to chemokine...

Bidentate PTP Inhibitors

Although pTyr is essential for peptide protein substrate recognition, pTyr alone does not possess high affinity for PTPs 45 . This and the fact that the PTP active site (pTyr binding site) is highly conserved among various PTPs present a serious challenge for the development of potent and selective PTP inhibitors targeted primarily to the active site. The discovery of a second aryl phosphate-binding site in PTP1B (defined by residues Arg24, Arg254, Met258, Gly259, and Gln262), which is not...

IgSuperfold Structures and Assemblies

The Ig-superfold is characterized by a primary sequence motif that spans some 100 amino acids. In three dimensions, this sequence motif translates into a compact domain structure that comprised of two anti-parallel P-sheets packed face to face (Fig. 1). Although there is a defined topology and connectivity for the Ig-superfold, the number of P-strands is variable. To take account of this variability Ig-like domains have been classified into different sets, according to the number and...

Mechanisms of GPCR Desensitization and Endocytosis

Urethrotoom

Heterotrimeric G Proteins Mediated by Receptor Phosphorylation Extensive studies of certain GPCRs, such as rhodopsin (a light-activated GPCR) and P2-AR (a ligand-activated GPCR), established a highly conserved mechanism that regulates the functional activity of many GPCRs 3-5,9 . This mechanism involves the phosphorylation of receptors by a specific family of G-protein-coupled receptor kinases (GRKs) followed by the interaction of phosphorylated receptors with cytoplasmic accessory proteins...

Cell Signaling Tomorrow

As the humane genome and importantly the genomes of several other key research paradigms reach completion in terms of sequence, interpretation, and full annotation, it will be possible to know, in a general sense, the complete complement of proteins, involved in cell signaling. Of course, this signaling proteome will contain a vast number of variants arising from message splicing and posttranslational modification. Appreciating how all of these variants interact as a function of time in...

Eph Receptor Signaling Via Cytoplasmic Protein Tyrosine Kinases

Upon ligand binding, Eph receptors are thought to dimer-ize, perhaps oligomerize, and autophosphorylate specific tyrosine residues of the cytoplasmic part of the partner receptor. Somewhat unusual among RTKs, Eph kinase activity is autoinhibited by the unphosphorylated juxtamembrane Figure 1 Eph receptor structure and cytoplasmic interactions. The intracellular part of Eph receptors consists of a juxtamembrane region, tyrosine kinase domain, SAM domain (of unknown function), and a PDZ binding...

High Affinity Variant of hGH hGHv Reveals an Altered Mode for Receptor Homodimerization

Using phage display mutagenesis selections, a variant of hGH that binds > 100-fold more tightly to hGH-R at site 1 was produced 41,42 . This variant (hGHv) has 15 mutations localized in its site 1 binding site and is fully biologically active but is totally specific to hGH-R, losing its ability to bind to hPRL-R. (This is a clinically relevant, second-generation hGH used for treating acromegaly). The recent high-resolution X-ray analysis of the ternary complex of hGHv bound to two copies of...

IRSProtein Structure and Function

Alignment of the amino acid sequences of the IRS-proteins reveals important similarities and subtle differences (Fig. 1). IRS 1-4 and Chico contain an NH2-terminal pleckstrin homology (PH) domain adjacent to a PTB domain. The structures of the PH and PTB domains are remarkably similar 30 , and both facilitate recruitment of IRS-proteins to the activated insulin and IGF1 receptors deletion of the PH and PTB domain almost completely prevents phosphoryla-tion of the C-terminus. The PTB domain...

The Transition State

In the transition state, noncovalent bonds are in the process of being made and broken. At least one encounter complex can be found prior to the transition state, with additional intermediates occupying the energy landscape past the transition state. What is the molecular basis of the transition state for protein-protein interactions The bound state of two proteins is characterized by local specific interactions (e.g., van der Waals, electrostatic) between widely desolvated binding sites,...

PP1 Regulatory or Targeting Subunits

Protein phosphatase 1 enzymes acquire specificity of function by their association with targeting regulatory pro teins that direct the enzyme to distinct subcellular structures or compartments in proximity to physiological substrates, confer substrate specificity and or modulate enzyme activity. Over 40 PPl-associated proteins are currently known. Most interact with PPlc through multiple sites, a shared site recognizable in many of the subunits, and other sites unique to the individual...

The Structure of the Atypical Kinase Domain Reveals Similarity to Classical Protein Kinases and to Metabolic Enzymes

The kinase domain of ChaK forms a dimer as a consequence of a domain-swapping exchange of an N-terminal 27-residue dimerization segment 17 . The isolated ChaK kinase domain is also a dimer in solution, and the dimeric property of ChaK may be relevant to the biological function or regulation of the kinase, as TRP channels, like other voltage-gated ion channels, are likely to be tetrameric. However, EF2 kinase and MHCK are monomers and would require an alternative extended polypeptide sequence to...

INAD Organizes Signaling Complexes

Drosophila Rh1 Phototransduction

Phototransduction in Drosophila is one of the fastest G-protein-coupled signaling pathways known, taking less than 20 ms from light activation to maximum response. This high speed of signaling is primarily due to the incorporation of signaling components into multi-protein complexes. Signaling complexes bring components into close proximity, promoting rapid interaction as well as ensuring the proper sub-cellular localization of components. The central organizer of these signaling complexes is...

Oxyanions as PTP Inhibitors

Inorganic phosphate is a hydrolytic product of the PTP reaction and serves as a reversible competitive PTP inhibitor K 5 mM 20 . Because of the similar physical and chemical properties, many oxyanions, including sulfate, arsenate, molybdate, and tungstate, can competitively inhibit PTPs to various degrees 10 M to millimolar levels . These oxyanions could inhibit PTPs by simply mimicking the tetrahedral geometry of the phosphate ion. The crystal structure of the PTP complexed with tungstate...

Ga Interactions with Effector Molecules

The specific effectors activated by Ga-GTP are dependent on the Ga subtype and are summarized in Table 1. Some properties of Ga-mediated effector activation deserve mention 1 Each Ga family activates a distinct profile of effectors. Co-crystallization of Gas with the catalytic domains of adenylyl cyclase AC has allowed the identification of specific contact sites within the subunit at the a2 helix and a3-P5 loop 44 . In addition to the Ga-GTP bound form the inactive form, Ga-GDP, can also...