Receptor binding and internalization of mouse interleukin-2 derivatives that are partial agonists.

Mouse interleukin-2 (mIL-2) mutant proteins with subunit-specific receptor binding defects have been previously described. Some of these mutant proteins are unable to trigger a maximum proliferative response of T cells. In this study, mIL-2 and mIL-2 mutant proteins were labeled with 32P, and their association and dissociation kinetics with the high affinity IL-2 receptor (IL-2R) were investigated. A mIL-2 mutant protein with a partial defect in binding to the low affinity component of IL-2R had a slower on-rate than mIL-2. On the other hand, a mIL-2 antagonist with a binding defect to the intermediate affinity component of IL-2R had a normal on-rate, whereas its off-rate at 37 degrees C was faster than mIL-2. This fast off-rate at physiological temperature interfered with mIL-2 internalization. When three mIL-2 partial agonists, each inducing a different maximal response, were examined, no difference was found between their dissociation rates or their internalization properties. The significance of these findings for the function of each receptor subunit in the IL-2R complex, as well as for the mechanism of activation of the receptor, is discussed.     

Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4.

DAB389-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-related fusion protein which has been shown to be selectively toxic to cells expressing the mIL-4 receptor. In this report, we have used site-directed and in-frame deletion mutagenesis to study the role of the putative C-terminal alpha-helix (helix E) of the mIL-4 component of DAB389-mIL-4 in the intoxication process. We demonstrate that deletion of the C-terminal 15 amino acids of the fusion toxin leads to loss of cytotoxicity. The substitution of Phe496 with either Pro, Ala or Tyr, results in a greater than 20-fold decrease in cytotoxic activity of the respective mutant fusion toxins. In addition, substitution of Leu497 with either Ala or Glu results in a similar loss of cytotoxic activity. All of these mutant forms of the mIL-4 fusion toxin demonstrate a significant decrease in binding affinity (Ki) to the mIL-4 receptor in a competitive radioligand binding assay. In marked contrast, however, the substitution of Asp495 with Asn results in a 4-fold increase in cytotoxic potency and binding affinity to mIL-4 receptor bearing cells in vitro.     

Discrete alterations of the insulin-like growth factor I molecule which alter its affinity for insulin-like growth factor-binding proteins result in changes in bioactivity.

Insulin-like growth factor (IGF)-binding proteins (BPs) bind IGF-I and IGF-II with high affinity. They are present in extracellular fluids and modulate the interactions of their ligands with the type 1 IGF cell surface receptor. These studies utilized IGF-I analogs that have reduced binding affinity for either the type 1 IGF receptor or binding proteins to study the ligand specificity of IGF-BP-1 and the role of IGF-BP-1 in modulating the biological activity of IGF-I. The data indicate that the regions of IGF-I which are responsible for binding to IGF-BP-1 and to human serum-binding proteins are distinct but overlapping and are clearly distinct from the type I receptor binding sites. In the absence of exogenously added IGF-BP-1, the analogs with reduced affinity for IGF-BP-1 are more potent than IGF-I in stimulating DNA synthesis by porcine aortic smooth muscle cells. In contrast, when cells are concomitantly exposed to IGF-BP-1, two of the analogs with reduced affinity for binding protein give only 40-65% of the maximal IGF-I response. [Leu24, 1-62]IGF-I, which has a 100-fold reduced affinity for the type 1 IGF receptor, gave a value that was 62% of the maximal IGF-BP-1 potentiated response. A second biological response, that of stimulating binding protein secretion by IGF-I, was also examined. [Leu24, 1-62]IGF-I is more potent than IGF-I whereas the activity of the analogs with lower affinity for IGF-BP-1 is significantly reduced. Thus, the ability to activate DNA synthesis and binding protein secretion maximally in the presence of IGF-BP-1 is dependent on the affinity of IGFs for both type 1 receptors and binding proteins.     

Receptor antagonist and selective agonist derivatives of mouse interleukin-2.

Mouse interleukin-2 (mIL-2) proteins with substitutions at two residues (D34 and Q141) that interact specifically with different signalling subunits (respectively, beta and gamma) of the IL-2 receptor (IL-2R) were examined using several in vitro cellular assays. Proteins with specific substitutions at both residues were partial agonists and their maximal responses varied widely in different IL-2-responsive cell types. Two of these cell types had comparable numbers of IL-2R and similar affinities for wild-type mIL-2 and mutant mIL-2 proteins. However, the more responsive cell type had 'spare' IL-2R. Various mIL-2 proteins with substitutions at Q141 had modest defects in IL-2R-binding and were potent antagonists of native mIL-2 action. Proteins with bulky or basic substitutions at residue D34 were weak antagonists due to severely reduced IL-2 binding and their reduced binding paralleled their defects in IL-2R activation. Our results suggest that interaction of mIL-2 with IL-2R beta is more important for binding than activation and that the converse holds for mIL-2 interaction with IL-2R gamma. Also genetic manipulation of the interaction of IL-2 with IL-2R beta and IL-2R gamma has led to the discovery of potentially useful IL-2 antagonists and selective agonists.     

Alteration of epidermal growth factor receptor activity by mutation of its primary carboxyl-terminal site of tyrosine self-phosphorylation

The epidermal growth factor (EGF) receptor, which exhibits intrinsic protein tyrosine kinase activity, undergoes a rapid, intramolecular self-phosphorylation reaction following EGF activation. The primary sites of tyrosine self-phosphorylation in vivo are located in the extreme carboxyl-terminal region of the molecule, principally Tyr-1173. To test the biological and biochemical consequences of this EGF receptor self-phosphorylation, we made the mutation Tyr----Phe-1173. Membranes containing the mutated receptor exhibited an ED50 for EGF activation of tyrosine kinase activity equivalent to control receptor at both high and low substrate levels, but exhibited reduced basal and EGF-stimulated tyrosine kinase activity at low, non-saturating substrate levels. The Tyr----Phe-1173 mutant possessed high affinity EGF binding and could still self-phosphorylate other tyrosine sites in an intramolecular fashion with a low Km for ATP (200 nM), suggesting that this alteration did not grossly change receptor structure. When EGF-dependent growth of Chinese hamster ovary cells expressing comparable levels of control or mutant EGF receptor was measured, the ability of the mutant receptor to mediate cell growth in response to EGF was reduced by approximately 50%, yet both receptors exhibited a similar affinity and ED50 for EGF. These results support the concept that this self-phosphorylation site can act as a competitive/alternate substrate for the EGF receptor, and that this region of the molecule is important in modulating its maximal biological activity.     

Molecular basis of a high affinity murine interleukin-5 receptor.

The mouse interleukin-5 receptor (mIL-5R) consists of two components one of which, the mIL-5R alpha-chain, binds mIL-5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL-5R beta-chain, immunoprecipitate a p130-140 protein doublet which corresponds to the mIL-3R and the mIL-3R-like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL-5R can be reconstituted on COS1 cells by co-expression of the mIL-5R alpha-chain with the mIL-3R-like protein (beta-chain). Cross-linking of 125I-labeled mIL-5 to the COS1 cells co-transfected with both cDNAs revealed the same pattern as in B13 cells, i.e. two proteins of 60 and 130 kd which correspond to the low affinity mIL-5R alpha-chain and the mIL-3R-like protein, respectively. The dissociation rate of mIL-5 from this reconstituted high affinity site was lower than that of the low affinity site, whereas the association rate was unchanged. Nonetheless, the apparent dissociation constant (Kd) for this reconstituted receptor was still 10-fold higher than the Kd observed for B13 cells. Although the mIL-3R is greater than 90% homologous to the mIL-3R-like protein, no increase in affinity for mIL-5 was detected on COS1 cells co-transfected with the cDNAs for the mIL-5R alpha-chain and the mIL-3R protein.     

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.     

Biochemical evidence for a third chain of the interleukin-2 receptor

Two receptor proteins that specifically bind interleukin-2 (IL-2) have been identified previously. The L (Tac or alpha) chain can bind IL-2 with a Kd value of 10 nM (low affinity). Although the H (beta) chain expressed on lymphocytes can bind IL-2 with a Kd value of 1 nM (intermediate affinity), transfected fibroblasts expressing the H chain cannot bind IL-2, suggesting the involvement of other lymphocyte-specific factors for the function of the H chain. To obtain direct evidence for the presence of a third component of the IL-2 receptor, we examined the IL-2 binding activity of detergent-solubilized cell membrane preparations. We found that lysates of transfected Cos7 cells expressing H chains can bind IL-2 when mixed with lysates from lymphocytes that cannot bind IL-2. Chemical cross-linking of 125I-IL-2-bound lysate mixture and subsequent immunoprecipitation with a noncompetitive anti-H chain antibody gave rise to two 125I-IL-2-bound proteins, a 56-kDa protein (p56) and the H chain, although neither the H chain nor p56 alone is able to bind IL-2. These results indicate that p56 is the IL-2 receptor third chain that is required for IL-2 binding to the H chain. A similar lysate mixing experiment also showed that p56 is involved in IL-2 binding to the high affinity IL-2 receptor by forming the quaternary complex of IL-2, p56, L chain, and H chain.     

Dispensability of glutamic acid 48 and aspartic acid 134 for Mn2+-dependent activity of Escherichia coli ribonuclease HI

The activities of the eight mutant proteins of Escherichia coli RNase HI, in which the four carboxylic amino acids (Asp(10), Glu(48), Asp(70), and Asp(134)) involved in catalysis are changed to Asn (Gln) or Ala, were examined in the presence of Mn(2+). Of these proteins, the E48A, E48Q, D134A, and D134N proteins exhibited the activity, indicating that Glu(48) and Asp(134) are dispensable for Mn(2+)-dependent activity. The maximal activities of the E48A and D134A proteins were comparable to that of the wild-type protein. However, unlike the wild-type protein, these mutant proteins exhibited the maximal activities in the presence of >100 microM MnCl(2), and their activities were not inhibited at higher Mn(2+) concentrations (up to 10 mM). The wild-type protein contains two Mn(2+) binding sites and is activated upon binding of one Mn(2+) ion at site 1 at low ( approximately 1 microM) Mn(2+) concentrations. This activity is attenuated upon binding of a second Mn(2+) ion at site 2 at high (>10 microM) Mn(2+) concentrations. The cleavage specificities of the mutant proteins, which were examined using oligomeric substrates at high Mn(2+) concentrations, were identical to that of the wild-type protein at low Mn(2+) concentrations but were different from that of the wild-type protein at high Mn(2+) concentrations. These results suggest that one Mn(2+) ion binds to the E48A, E48Q, D134A, and D134N proteins at site 1 or a nearby site with weaker affinities. The binding analyses of the Mn(2+) ion to these proteins in the absence of the substrate support this hypothesis. When Mn(2+) ion is used as a metal cofactor, the Mn(2+) ion itself, instead of Glu(48) and Asp(134), probably holds water molecules required for activity.     

Expression and in vitro properties of guinea pig IL-5: comparison to human and murine orthologs

Interleukin-5 (IL-5) is a key mediator of eosinophilic inflammation. The biological role of this cytokine in an allergic airway inflammatory response has been widely demonstrated in guinea pigs, yet the interaction of guinea pig IL-5 (gpIL-5) with its receptor has not been studied. Experiments were performed to quantitate the interaction of gpIL-5 with gpIL-5r and to compare this affinity with that of hIL-5 and mIL-5 and their cognate receptors. The cross-species affinity and agonist efficacy were evaluated to see if gpIL-5r had a restricted species reactivity (as is the case with mIL-5r) or did not distinguish between IL-5 orthologs (similar to hIL-5r). gpIL-5 was cloned using mRNA isolated from cells obtained by bronchoalveolar lavage. Recombinant gpIL-5 was expressed in T. ni insect cells and purified from spent media. Binding assays were performed using insect cells expressing hIL-5ralphabeta or gpIL-5ralphabeta1 as previously described (Cytokine, 12:858-866, 2000) or using B13 cells which express mIL-5r. The agonist potency and efficacy properties of each IL-5 ortholog were evaluated by quantitating the proliferative response of human TF-1 cells and murine B13 cells. gpIL-5 bound with high affinity to recombinant gpIL-5r as demonstrated by displacing [125I]hIL-5 (Ki = 160 pM). gpIL-5 also bound to hIL-5r with high affinity (Ki = 750 pM). hIL-5 and mIL-5 showed similar, high-affinity binding profiles to both gpIL-5r and hIL-5r. In contrast, gpIL-5 and hIL-5 did not bind to the mIL-5r as demonstrated by an inability to displace [125I]mIL-5, even at 1000-fold molar excess. These differences in affinity for IL-5r orthologs correlated with bioassay results: human TF-1 cells showed roughly comparable proliferative responses to guinea pig, human and murine IL-5 whereas murine B13 cells showed a strong preference for murine over guinea pig and human IL-5 (EC50 = 1.9, 2200 and 720 pM, respectively). Recombinant gpIL-5 binds to the gpIL-5r with high affinity, similar to that seen with the human ligand-receptor pair. gpIL-5r and hIL-5r do not distinguish between the three IL-5 orthologs whereas mIL-5r has restricted specificity for its cognate ligand.     

Definition and spatial location of mouse interleukin-2 residues that interact with its heterotrimeric receptor.

The high affinity receptor for interleukin-2 (IL-2) contains three subunits called IL-2R alpha, beta and gamma. A biological and receptor binding analysis based on 1393 different mutant mouse IL-2 (mIL-2) proteins was used to define the function of each of the 149 residues. By this genetic analysis, 44 residues were assigned important functions, 21 of which were structural. The remaining 23 residues consisted of 19 residues, from three separate regions, that were important for IL-2R alpha interaction; three residues, from two separate regions, that were important for IL-2R beta interaction; and a single residue important for IL-2R gamma interaction. We built a model mIL-2 structure based on the homologous human IL-2 (hIL-2) crystal structure. The roles of the 21 residues presumed to be important for structure were consistent with the model. Despite discontinuity in the primary sequence, the residues specific for each IL-2R subunit interaction were clustered and located to three disparate regions of the tertiary mIL-2 structure. The relative spatial locations of these three surfaces are different from the two receptor binding sites known for the structurally related human growth hormone and the significance of this observation is discussed.     

Biological characterization of human interleukin-2 mutant proteins. Structure-activity relationship studies

Several human interleukin-2 (IL-2) mutant proteins have been produced previously by site-directed mutagenesis and found to have different capacities to induce T-cell proliferative activity. In this study, the abilities of these IL-2 mutant proteins to activate natural killer cells and to induce interferon-gamma production have been evaluated, and the binding of these proteins to IL-2 receptors analyzed. Natural killer cell activation and interferon-gamma induction assays showed that the relative activities of IL-2 mutant proteins were consistent with their relative activities in T-cell proliferation assay. Receptor-binding studies showed that the activities of most proteins correlated well with their respective affinities for high-affinity IL-2 receptors on CTLL-2 cells. Interestingly, although the mutant protein with deletion of cysteine 125 (des-Cys125) was biologically less active than the protein with substitution of alanine for cysteine 105 (Ala105), both proteins exhibited similar affinity. Des-Cys125, like IL-2 and Ala105, also caused down-regulation of high-affinity IL-2 receptors. Binding studies on MLA-144, a cell line expressing mainly intermediate-affinity IL-2 receptors (IL-2R beta), however, showed that des-Cys125 had much lower affinity than Ala105. These results suggest that binding of IL-2 and mutant proteins to the IL-2R beta component of the high-affinity receptor is essential for the induction of biological effects.     

Production and characterisation of Met80X mutants of yeast iso-1-cytochrome c: spectral,photochemical and binding studies on the ferrous derivatives

The iron ligand, Met80, of yeast iso-1-cytochrome c has been mutated to residues that are unable to bind to the iron. The resultant proteins, Met80Ala, Ser, Asp, Glu, have been expressed and purified. All mutant proteins exhibit well defined pH dependent spectral transitions that report the binding, at high pH, of an intrinsic ligand (probably the nitrogen of an epsilon-NH(2) of a lysine) that drives the heme low-spin. The pK values are mutant dependent. All the mutant proteins bind extrinsic ligands, such as CO, in their ferrous states and we report the apparent quantum yield (phi) for CO photo-dissociation. The values of phi range from 0.004 for Met80Ala to 0.04 for Met80Asp. We also report values for the rate constant for binding the intrinsic lysine residue. The values for this constant, for phi and for the pK values are discussed in terms of the rigidity of the cytochrome structure. We also show that the mutant proteins bind with high affinity to cytochrome c oxidase, both in the ferric and ferrous states. The potential of these proteins to act as light activated electron donors for the study of electron transfer is discussed.     

Biochemical analysis of yeast G(alpha) mutants that enhance adaptation to pheromone

The mating-specific heterotrimeric G(alpha) protein of Saccharomyces cerevisiae, Gpa1, negatively regulates activation of the pheromone response pathway both by sequestering G(beta)gamma and by triggering an adaptive response through an as yet unknown mechanism. Previous genetic studies identified mutant alleles of GPA1 that downregulate the pheromone response independently of the pheromone receptor (GPA1E364K), or through a receptor-dependent mechanism (GPA1N388D). To further our understanding of the mechanism of action of these mutant alleles, their corresponding proteins were purified and subjected to biochemical analysis. The receptor-dependent activity of Gpa1N388D was further analyzed using yeast strains expressing constitutively active receptor (Ste2) mutants, and C-terminal truncation mutant forms of Gpa1. A combination of G(alpha) affinity chromatography, GTP binding/hydrolysis studies, and genetic analysis allowed us to assign a distinct mechanism of action to each of these mutant proteins.     

A mutation of the beta 2-adrenergic receptor impairs agonist activation of adenylyl cyclase without affecting high affinity agonist binding. Distinct molecular determinants of the receptor are involved in physical coupling to and functional activation of Gs

Activation of guanyl nucleotide regulatory proteins (G proteins) by hormones and neurotransmitters appears to require the formation of high affinity agonist-receptor-G protein ternary complexes. In the case of the beta 2-adrenergic receptor, multiple regions of the molecule have been implicated in coupling to the stimulatory G protein Gs. This finding raises the possibility that discrete regions of the receptor mediate ternary complex formation, whereas different loci may be involved in other aspects of G protein activation. To date, however, mutagenesis studies with the beta 2-adrenergic receptor have not clarified this question since mutant receptors with impaired abilities to activate Gs have generally possessed a diminished capacity to form the ternary complex as assessed in binding assays. We have expressed in a mammalian cell line a mutant beta 2-adrenergic receptor comprising a seven-amino acid deletion in the carboxyl-terminal region of its third cytoplasmic loop (D267-273), a region proposed to be critically involved in coupling to Gs. When tested with beta-adrenergic agonists, the maximal adenylyl cyclase response mediated by this mutant receptor was less than one-half of that seen with the wild-type receptor. Nevertheless, D267-273 exhibited high affinity agonist binding identical to that of the wild-type receptor. In addition, agonist-induced sequestration of the receptor, a property not mediated by Gs, was also normal. These findings indicate that the formation of high affinity agonist-receptor-Gs complexes is not sufficient to fully activate Gs. Instead, an additional stimulatory signal appears to be required from the receptor. Our data thereby suggest that the molecular determinants of the beta 2-adrenergic receptor involved in formation of the ternary complex are not identical to those that transmit the agonist-induced stimulatory signal to Gs.     

Identification of three adjacent amino acids of interleukin-2 receptor beta chain which control the affinity and the specificity of the interaction with interleukin-2.

The beta chain of the interleukin-2 (IL-2) receptor (IL-2R beta) and the interleukin-3 (IL-3) binding protein AIC2A are members of the family of cytokine receptors, which also includes the receptors for growth hormone (GHR) and prolactin. A four amino acid sequence of AIC2A has recently been shown to be critical for IL-3 binding. We analyze here the function of the analogous sequence of human IL-2R beta and identify three amino acids, Ser132, His133 and Tyr134, which play a critical role in IL-2 binding. We show that some mutant IL-2 proteins with substitutions of a critical Asp residue in the N-terminal alpha-helix bind the mutant IL-2R beta receptor with a higher affinity than the wild-type receptor. This suggests that the critical Asp34 in the ligand and the sequence Ser-His-Tyr (positions 132-134) in the receptor interact directly. On the double barrel beta-stranded structural model of cytokine receptors, the residues important for ligand binding in IL-2R beta, AIC2A and GHR map to strikingly similar locations within a barrel, with the interesting difference that it is the N-terminal barrel for GHR and the C-terminal barrel for IL-2R beta and AIC2A.     

An insertional mutant of epidermal growth factor receptor allows dissection of diverse receptor functions.

Cultured NIH-3T3 cells devoid of endogenous EGF-receptors were transfected with cDNA constructs encoding normal human EGF-receptor and with a construct encoding an insertional mutant of the EGF-receptor containing four additional amino acids in the kinase domain after residue 708. Unlike the wild-type receptor expressed in these cells which exhibits EGF-stimulatable protein tyrosine kinase activity, the mutant receptor lacks protein tyrosine kinase activity both in vitro and in vivo. Despite this deficiency the mutant receptor is properly processed, it binds EGF and it exhibits both high and low affinity binding sites. Moreover, it undergoes efficient EGF-mediated endocytosis. However, EGF fails to stimulate DNA synthesis and is unable to stimulate the phosphorylation of S6 ribosomal protein in cells expressing this receptor mutant. Hence, it is proposed that the protein tyrosine kinase activity of EGF-receptor is essential for the initiation of S6 phosphorylation and for DNA synthesis induced by EGF. However, EGF-receptor processing, the expression of high and low affinity surface receptors and receptor internalization, require neither kinase activity nor receptor autophosphorylation. Interestingly, phorbol ester (TPA) fails to abolish the high affinity state and is also unable to stimulate the phosphorylation of this receptor mutant. This result is consistent with the notion that kinase-C phosphorylation of EGF-receptor is essential for the loss of high affinity EGF-receptors caused by TPA.     

Calcium-binding EGF-like modules in coagulation proteinases: function of the calcium ion in module interactions

Epidermal growth factor (EGF)-like modules are involved in protein-protein interactions and are found in numerous extracellular proteins and membrane proteins. Among these proteins are enzymes involved in blood coagulation, fibrinolysis and the complement system as well as matrix proteins and cell surface receptors such as the EGF precursor, the low density lipoprotein receptor and the developmentally important receptor, Notch. The coagulation enzymes, factors VII, IX and X and protein C, all have two EGF-like modules, whereas the cofactor of activated protein C, protein S, has four EGF-like modules in tandem. Certain of the cell surface receptors have numerous EGF modules in tandem. A subset of EGF modules bind one Ca(2+). The Ca(2+)-binding sequence motif is coupled to a sequence motif that brings about beta-hydroxylation of a particular Asp/Asn residue. Ca(2+)-binding to an EGF module is important to orient neighboring modules relative to each other in a manner that is required for biological activity. The Ca(2+) affinity of an EGF module is often influenced by its N-terminal neighbor, be it another EGF module or a module of another type. This can result in an increase in Ca(2+) affinity of several orders of magnitude. Point mutations in EGF modules that involve amino acids which are Ca(2+) ligands result in the biosynthesis of biologically inactive proteins. Such mutations have been identified, for instance, in factor IX, causing hemophilia B, in fibrillin, causing Marfan syndrome, and in the low density lipoprotein receptor, causing hypercholesterolemia. In this review the emphasis will be on the coagulation factors.     

Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding.

Recent studies on the glucagon antagonist des-His1-[Glu9]glucagon amide have resulted in pure inhibitors of the hormone, suggesting that the inhibitory properties may be centered around position 9. The present study was designed to investigate the chemical characteristics of substitutions in position 9 of glucagon that determine binding affinity and biological activity. Twenty replacement analogs of position 9 of glucagon were synthesized and assessed for their ability to bind to the glucagon receptor in rat hepatocyte membranes and to activate adenylate cyclase. Any substitution of aspartic acid 9 was accompanied by a severely diminished capacity to transmit the biological signal, while retaining receptor binding affinity. These results are an indication of an uncoupling of receptor binding and biological activity at this locus and define a central role of aspartic acid 9 in glucagon activity. Single replacement or deletion of either His1 or Asp9 in glucagon caused a 20- to 50-fold decrease in cyclase activity, whereas these same changes made in tandem caused virtually complete loss of activity, with decreases of 10(4)-to 10(6)-fold. These observations have led us to speculate that, at the molecular level, the region of glucagon required for transduction of the biological response may be distinct from the binding region and is mediated by a coupled interaction between His1 and Asp9 of the hormone and a complementary functional site of the glucagon receptor.