Molecular properties of the oxytocin/bovine neurophysin biosynthetic precursor. Studies using a semisynthetic precursor

An oxytocin/bovine neurophysin I biosynthetic precursor, [N epsilon-diacetimidyl-30,71, des-His106]pro-OT/BNPI, was synthesized from a synthetic oxytocinyl peptide, 1/2Cys-Tyr-Ile-Gln-Asn-1/2Cys-Pro-Leu-Gly-Gly-Lys-Arg, and native neurophysin by chemical semisynthesis. The semisynthetic precursor contains the entire sequence of the biosynthetic precursor deduced from the complementary DNA structure except for omission of the carboxyl-terminal histidine residue. The covalent structure of the semisynthetic product was verified by amino acid analysis and amino-terminal analysis. Analytical affinity chromatography was employed to evaluate noncovalent binding properties of the precursor. The precursor does not bind significantly to immobilized Met-Tyr-Phe, a hormone binding site ligand. In contrast, the acetimidated precursor binds to immobilized bovine neurophysin II, with a 13-fold higher affinity than does acetimidated neurophysin itself. When a hormonal ligand, [Lys8]vasopressin, was added to the elution buffer at the concentration of 0.1 mM so that a major portion of the immobilized BNPII was liganded, the affinity between the immobilized liganded BNPII and the precursor was enhanced 8-fold and approached the affinity for the liganded (bovine neurophysin I-immobilized BNPII) interaction. The data imply that the precursor can self-associate and that this self-association is closely related to that of liganded neurophysin. The tripeptide affinity matrix data argue that, in the precursor, the ligand binding site of the neurophysin domain is occupied intramolecularly by the hormone domain. The data verify the view that both the self-association surface and hormone binding site are established upon precursor folding. A disulfide stability analysis showed the resistance, to disulfide interchange by dithiothreitol, of semisynthetic precursor but not of neurophysin, as judged by protein association and peptide ligand binding activities, respectively. The results argue that the molecular structure of the precursor is established upon precursor folding and before enzymatic processing that produces mature hormone and neurophysin.     

Structural requirements of peptide hormone binding for peptide-potentiated self-association of bovine neurophysin II

Site-specific, truncated, and sequence-simplified analogs of the hormone [Arg8]vasopressin were investigated for the relationship between their abilities to recognize immobilized bovine neurophysin and to promote neurophysin self-association. Peptide binding to neurophysin was measured quantitatively by analytical high performance affinity chromatography on immobilized bovine neurophysin II. Neurophysin self-association, measured as binding of soluble to immobilized neurophysin, was promoted (made higher affinity) by soluble peptide hormone and its analogs, with the effect of particular peptides being proportional to their binding affinities for neurophysin. Sequence-redesigned peptides able to recognize neurophysin, including dipeptide amides, were able to potentiate the self-association to the same extent as the natural hormone when tested at concentrations adjusted to effect equal degrees of saturation of neurophysin. The relationship between peptide affinity to neurophysin and the potentiation of self-association suggests that the latter is directly dependent on the former and can occur even with limited segments of hormone sequence. The data fit best to a model in which hormone binding and self-association surfaces of neurophysin are separate and linked through the neurophysin molecule to produce cooperativity (hormone-promoted self-association). Given that only limited structural elements of hormone are required for promoting self-association, the results fit less well with models in which cooperativity requires that hormone make dimer-stabilizing contacts with both self-associating subunits of neurophysin simultaneously.     

Effect of neurophysin on enzymatic maturation of oxytocin from its precursor

We examined the extent to which rates of enzymatic conversion of the oxytocin biosynthetic precursor to mature peptide are modulated by intramolecular and intermolecular assembly of precursor and polypeptide intermediates. The biosynthesized precursor contains hormone and neurophysin sequences linked by a Gly-Lys-Arg sequence and undergoes enzymatic processing reactions which include endoproteolytic cleavage at the Lys-Arg dibasic sequence, carboxypeptidase B-like exoproteolytic cleavage, and enzymatic amidation. We evaluated the effect of neurophysin on such processing reactions using semisynthetic precursors of oxytocin/bovine neurophysin I and synthetic oxytocinyl precursor intermediates as substrates. Neurophysin I at high concentration (0.7 mM) reduced the rates of carboxy-peptidase B-like conversion of oxytocinyl-Gly-Lys-Arg to oxytocinyl-Gly and the enzymatic amidation of oxytocinyl-Gly to mature (C-terminal amidated) oxytocin. The dependence of rate suppression on the concentrations of peptide substrate and neurophysin I suggested that suppression is due to intermolecular formation of hormone-neurophysin complexes which are aggregated at least to dimers. An analogous intramolecular neurophysin effect was found for endoproteolytic processing of semisynthetic precursors. Endoproteinase Lys-C cleaved the Lys11-Arg12 peptide bond in a native-like semisynthetic precursor at a significantly slower rate than it did an assembly-deficient precursor analogue. The difference in semisynthetic precursor endoproteolysis rates is most substantial at the high concentrations at which the native-like precursor would form dimers but the assembly-deficient analogue would not. The native-like semisynthetic precursor was more stable than the assembly-deficient precursor analogue to tryptic digestion. The concentration-dependent effects of neurophysin, both intramolecularly as a precursor domain and intermolecularly as an interacting protein, are likely to occur in the secretory granules in which the biosynthetic precursors are packaged. The molecular organization of both hormone/neurophysin precursors and the noncovalently complexed hormone-neurophysin intermediates can be expected to play a role in modulating enzymatic processing reactions that lead to mature neurohypophysial hormones.     

Proocytocin/neurophysin convertase from bovine neurohypophysis and corpus luteum secretory granules: complete purification, structure-function relationships, and competitive inhibitor

Structure-function relationship studies were conducted on the proocytocin/neurophysin endoprotease previously characterized in both bovine neurohypophyseal and corpus luteum granules, using as a reference substrate a synthetic peptide reproducing the entire (1-20) NH2-terminal domain of the precursor. The [D-Arg12] derivative of proocytocin/neurophysin (1-20) was found to be a good competitive inhibitor of the enzyme (Ki = 30 microM), while the [D-Lys11] derivative was not. This allowed the complete purification of two isoforms of the endoprotease (Mr 58,000 and 52,000, respectively) by affinity chromatography using covalently immobilized [D-Arg12] proocytocin/neurophysin (1-20) as the affinity adsorbent. The use of selectively modified or truncated forms of the reference substrate or of the [D-Arg12] competitive inhibitor of the endoprotease established clearly that this basic pair specific convertase is sensitive to modification of the substrate structure either at the basic residues of the cleavage locus or at amino acids around this site (i.e., Pro7 and Gly9). It is concluded that longer distance interactions between amino acids situated on both the NH2 and COOH sides of the basic doublet Lys11Arg12 may contribute to the stabilization of a preferred substrate conformation allowing recognition by the enzyme subsites.     

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.     

Thyrotropin-releasing hormone binding to the mouse pituitary receptor does not involve ionic interactions. A model for neutral peptide binding to G protein-coupled receptors.

Thyrotropin-releasing hormone, TRH (< Glu-His-Proamide), and [N tau-Me-His]TRH (MeTRH) are present as neutral and positively charged forms at physiologic pH, and it was possible that they bind to the TRH receptor (TRH-R) as charged (protonated) species. Binding affinities of TRH and MeTRH to endogenous rat TRH-Rs and to transfected wild type mouse TRH-Rs decreased below pH 7.1. Half-maximal decreases in binding occurred at the approximate pK alpha values of these ligands. Asp to Ala mutations in extracellular loop 1, TM-4, and TM-5 did not decrease binding affinity, but an Asp to Ala mutation in TM-2 caused the affinity to decrease 8-fold. The pH dependences of binding of MeTRH, however, were similar in wild type and all mutant receptors and were consistent with the protonated form of MeTRH binding less well. Thus, the binding of TRH to its receptor does not involve ionic interactions and may be a prototype for binding of neutral peptide ligands to G protein-coupled receptors.     

Expression, folding, and thermodynamic properties of the bovine oxytocin-neurophysin precursor: relationships to the intermolecular oxytocin-neurophysin complex.

Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor. To address this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed in Escherichia coli and folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented. The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor. Consistent with this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding between oxytocin and neurophysin in the precursor had only a small thermodynamic advantage over the corresponding bonding in the intermolecular complex. Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10. Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein.     

Partial purification and functional properties of an endoprotease from bovine neurosecretory granules cleaving proocytocin/neurophysin peptides at the basic amino acid doublet

An enriched preparation of neurosecretory granules from bovine pituitary neural lobes was used as a source of processing enzymes possibly involved in the cleavage of the proocytocin/neurophysin precursor. A synthetic eicosapeptide reproducing the entire (1-20) sequence of the NH2-terminal domain of the bovine ocytocin/neurophysin precursor was used as a substrate to monitor an endoprotease activity cleaving at the Lys11-Arg12 doublet. The 58-kDa endoprotease detected in the lysate of neurohypophyseal granules produced a single cleavage, after the doublet, at the Arg12-Ala13 peptide bond. This endoprotease with pHi 6.9 and 7.2 exhibits maximal activity at pH around neutrality (7.0) and was strongly inhibited by divalent cation chelating agents [ethylenediaminetetraacetic acid and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',-N'-tetraacetic acid] and to some extent by p-(chloromercuri)benzoate and p-(chloromercuri)benzenesulfonic acid, while phenylmethanesulfonyl fluoride and pepstatin were not active. This endoprotease action was sensitive to any modification of the substrate at either basic amino acid of the doublet since replacement of either L-Lys11 or L-Arg12 by D-Lys or D-Arg and by L-Nle abolished the cleavage reaction. In contrast, reversal of the polarity of the doublet in [Arg11,Lys12]proocytocin/neurophysin(1-20) had no effect on the mode of endoproteolytic cleavage as well as modifications of Gly10 (replaced by Ala10). It is concluded that the selectivity of this endoprotease, which may be involved in the primary event occurring in proocytocin/neurophysin processing, is strictly dependent upon the integrity of the basic doublet but that other parameters determined by the amino acid sequence around this doublet may play an important role.     

Formation of a native-like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor.

In the folding of bovine pancreatic trypsin inhibitor (BPTI), the single-disulfide intermediate [30-51] plays a key role. We have investigated a recombinant analog of [30-51] using a 2-dimensional nuclear magnetic resonance (2D-NMR). This recombinant analog, named [30-51]Ala, contains a disulfide bond between Cys-30 and Cys-51, but contains alanine in place of the other cysteines in BPTI to prevent the formation of other intermediates. By 2D-NMR, [30-51]Ala consists of 2 regions-one folded and one predominantly unfolded. The folded region resembles a previously characterized peptide model of [30-51], named P alpha P beta, that contains a native-like subdomain with tertiary packing. The unfolded region includes the first 14 N-terminal residues of [30-51] and is as unfolded as an isolated peptide containing these residues. Using protein dissection, we demonstrate that the folded and unfolded regions of [30-51]Ala are structurally independent. The partially folded structure of [30-51]Ala explains many of the properties of authentic [30-51] in the folding pathway of BPTI. Moreover, direct structural characterization of [30-51]Ala has revealed that a crucial step in the folding pathway of BPTI coincides with the formation of a native-like subdomain, supporting models for protein folding that emphasize the formation of cooperatively folded subdomains.     

Role of the streptokinase alpha-domain in the interactions of streptokinase with plasminogen and plasmin

The role of the streptokinase (SK) alpha-domain in plasminogen (Pg) and plasmin (Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [Glu]Pg, [Lys]Pg, and [Lys]Pm, and the SK truncation mutants, SK-(55-414), SK-(70-414), and SK-(152-414). Lysine binding site (LBS)-dependent and -independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-(1-54). The affinity of SK for [Glu]Pg was LBS-independent, and although [Lys]Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [Glu]Pg 2-7-fold and [Lys]Pg 相似文献   

Efficient folding of the FcepsilonRI alpha-chain membrane-proximal domain D2 depends on the presence of the N-terminal domain D1

Human high affinity receptor for IgE is a membrane glycoprotein multichain complex presenting two extracellular Ig modules in its alpha-chain (D1D2). The receptor IgE binding region is located within the membrane-proximal module D2, while the N-terminal module D1 appears to promote an optimal receptor conformation for IgE binding. To understand the structural relationship between the two modules, we dissected FcepsilonRI alpha-chain into its discrete Ig units and expressed them in mammalian cells. Unexpectedly, D2 was secreted as a disulphide-linked dimer, while D1 was monomeric. Active secretion and full glycosylation of dimeric D2 suggest a native-like conformation of the protein, justifying the escape from the endoplasmic reticulum/Golgi quality control systems. We then propose a domain-swapping model for D2, in which two interdigitated polypeptide chains assume the overall conformation of two Ig modules, as observed for rat CD2 N-terminal domain. Fusion of an unrelated Ig fold moiety at the N terminus of D2 did not interfere with its dimerisation. While D1D2 assumes a correct fold, co-expression of both isolated domains in the same cell did not restore monomeric folding of D2. Thus, D1 appears to assist the appropriate folding of FcepsilonRI alpha-chain, acting as an uncleavable intramolecular chaperone-like block towards D2.     

The relation of predicted structure to observed conformation and activity of glucagon analogs containing replacements at positions 19, 22, and 23

Six new analogs of glucagon have been synthesized containing replacements at positions 19, 22, and 23. They were designed to study the correlation between predicted conformation in the 19-27 segment of the hormone and the conformation calculated from circular dichroism measurements and the observed activation of adenylate cyclase in the liver membrane. The analogs were [Val19]glucagon, [Val22]glucagon, [Glu23]glucagon, [Val19,Glu23]glucagon, [Glu22,Glu23]glucagon, and [Ala22,Ala23]glucagon. The structures predicted for the 19-27 segment ranged from strongly alpha helical to weakly beta sheet. The observed conformations varied as functions of amino acid composition, solvent, concentration, pH, and temperature but did not correlate well with prediction. There was, however, a correlation between predicted structure and activation of adenylate cyclase in rat liver membranes.     

Recognition properties of antisense peptides to Arg8-vasopressin/bovine neurophysin II biosynthetic precursor sequences

We studied the interaction properties of synthetic antisense (AS) peptides encoded in the antisense strand of DNA corresponding to the N-terminal 20-residue sequence of the biosynthetic precursor of Arg8-vasopressin (AVP) and its binding protein bovine neurophysin II (BNPII). Binding affinities of sense polypeptides AVP and BNPII with AS peptides were measured by analytical affinity chromatography, in each case by the extent of chromatographic retardation of a soluble polypeptide interactor on an affinity matrix containing the other interactor as the immobilized species. Chromatographically calculated dissociation constants ranged from 10(-3) to 10(-6) M. Experiments were carried out to define the selectivity and underlying forces involved in the AS peptide interactions. For AS peptide elutions on sense peptide affinity supports, reduced binding affinity with increasing 1-propanol concentration and ionic strength suggested the presence of both ionic and hydrophobic contributions to AS peptide/immobilized sense peptide recognition. This same conclusion was reached with the antisense peptides as the immobilized species and measurement of elution of sequence-simplified, truncated, and charge-depleted forms of sense peptides. Immobilized AS 20-mer affinity matrix differentially retarded AVP versus oxytocin (OT) and BNPII versus BNPI (the neurophysin related biosynthetically to OT) and was used to separate these polypeptides from acid extracts of bovine posterior pituitaries. In addition, immobilized AS 12-mer corresponding to AVP-Gly-Lys-Arg could be used to separate AVP from OT. The results confirm that antisense peptides recognize sense peptides with significant selectivity in the AVP/BNPII precursor case.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonism by GRP(18-27) and substance P analogues on insulin release stimulated by GRP(18-27).

The antagonistic effects of [D-Phe25]gastrin-releasing peptide (GRP)(18-27) and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P (SP) on the stimulation of insulin release by GRP(18-27) from isolated canine pancreas were compared with that of [Ala23]GRP(18-27). The stimulation of insulin release by 1 nM GRP(18-27) was reduced to 24.1% and 15.4% by the prior infusion of 1 microM of [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP and 10 microM of [D-Phe25]GRP(18-27), respectively. Glucagon release by GRP(18-27) was not affected by these peptides using the above concentrations. The results indicate that these peptides are antagonists of bombesin-like peptide receptors on pancreatic B-cells, although the inhibitory activities are lower than that of [Ala23]GRP(18-27).     

Evidence for beta-turn structure in model peptides reproducing pro-ocytocin/neurophysin proteolytic processing site

The structural organization of small peptides reproducing the amino acid sequence of the common ocytocin/neurophysin precursor around the LysArg cleavage locus was investigated by a combination of spectroscopical techniques. In water both circular dichroism and [1H] NMR spectra indicated that these peptides adopted a random conformation. Evidence for folded structures was obtained when these compounds were placed in a membrane-like environment i.e. 40 mM SDS in phosphate buffer or trifluoroethanol. Whereas the CD spectra indicated the formation of various types of beta-turn in rapid equilibrium, measurements of NH temperature coefficients and Nuclear Overhauser Effects by 400 and 500 MHz NMR revealed the existence of contacts and of a folded conformation. These observations are discussed in relation with previous hypothesis made on the secondary structure organization of the proteolytic processing site of polypeptide hormone precursors.     

Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation

The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.     

Synthesis and biological activity of dynorphin-(1-13) and analogs substituted in positions 8 and 10

Dynorphin-(1-13) (Dyn-(1-13)) and various analogs substituted in positions 8 and 10 were synthesized by the solid-phase technique and analyzed for their ability to inhibit the electrically evoked contraction of the guinea pig ileum (GPI) and to compete with the binding of [3H]-ethylketocyclazocine (EKC, kappa ligand), [3H]-[D-Ala2, MePhe4-Gly-ol5]-enkephalin (DAGO, mu ligand) and [3H]-[D-Ser2, Thr6]-Leu-enkephalin (DSLET, delta ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Introduction of Ala in position 8 decreased the activity of the peptide on the GPI by 50% but induced a 2.22-fold increase in its affinity for the kappa receptor ([3H]-EKC binding displacement from guinea pig cerebellum; Ki of 0.05 nM as compared with 0.11 nM for Dyn-(1-13)). On the other hand, the ability of [Ala8] Dyn-(1-13) to displace the binding of [3H]-DSLET from rat brain membranes was decreased by a factor of 1.7 while its affinity for the mu receptor was not greatly affected ([3H]-DAGO displacement; Ki of 0.44 nM as compared with 0.50 nM for Dyn-(1-13)). Replacement of position 8 by D-Ala caused similar changes in the activity of the peptide but the increase in its affinity for the kappa site was somewhat smaller (Ki of 0.08 nM as compared with 0.11 nM). [D-Pro10]-Dyn-(1-13) was equipotent to [Ala8]-Dyn-(1-13) in the GPI but its affinity for the mu binding site was decreased by a factor of 2.7 as compared with Dyn-(1-13).(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of a single residue changes the binding properties of the serotonin 5-HT2 receptor from a human to a rat pharmacology.

Mesulergine displays approximately 50-fold higher affinity for the rat 5-HT2 receptor than for the human receptor. Comparison of the deduced amino acid sequences of cDNA clones encoding the human and rat 5-HT2 receptors reveals only 3 amino acid differences in their transmembrane domains. Only one of these differences (Ser----Ala at position 242 of TM5) is near to regions implicated in ligand binding by G protein-coupled receptors. We investigated the effect of mutating Ser242 of the human 5-HT2 receptor to an Ala residue as is found in the rat clone. Both [3H]mesulergine binding and mesulergine competition of [3H]ketanserin binding showed high affinity for rat membranes and the mutant human clone but low affinity for the native human clone, in agreement with previous studies of human postmortem tissue. These studies suggest that a single naturally occurring amino acid change between the human and the rat 5-HT2 receptors makes a major contribution to their pharmacological differences.     

The composition of crystalline complexes of neurophysin-M with [8-arginine]-vasopressin and oxytocin

Neurophysin-M, a methionine-containing protein that is the major constituent of neurophysin, has been crystallized as complexes with [8-arginine]-vasopressin. Three moles of vasopressin alone or 2 moles of vasopressin together with 1 mole of oxytocin are bound/mole of protein. An amorphous complex of the protein with oxytocin alone contains 2 moles of the hormone/mole of protein. Deamino-[8-arginine]-vasopressin, a highly active basic analogue of vasopressin, is not bound by neurophysin. The primary amino group of both vasopressin and oxytocin is necessary for binding with neurophysin.     

Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1,Val1,Asn2, Gln3,His4,Ser8, His9,Glu12,Tyr15,Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has greater than 1,000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3,Ala4]IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15,Leu16]IGF-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. This peptide is also equipotent to hIGF-I at the types 1 and 2 IGF receptors. The peptide in which these four-point mutations are combined, [Gln3,Ala4,Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. This peptide has 10-fold increased potency for the insulin receptor, but is equipotent to hIGF-I at the types 1 and 2 IGF receptors. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, these peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.