Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains. Roles of the CGLCG motif in the D1- and D3-domains

Plasmids encoding the amino-terminal region of porcine submaxillary mucin were modified by site-specific mutagenesis to assess the roles of individual half-cystine residues in the assembly of disulfide-linked multimers of mucin. COS-7 cells with the plasmid containing C1199A expressed primarily monomers, suggesting that half-cystine 1199 in the D3-domain is involved in forming mucin multimers. This residue is in the sequence C1199SWRYEPCG, which is highly conserved in the D3-domain of other secreted mucins and human prepro-von Willebrand factor. In contrast, cells with the plasmid containing C1276A expressed trimers like those with unmutated plasmid, suggesting that half-cystine 1276 is not involved in formation of disulfide-bonded multimers. The roles of the half-cystines in the CGLCG motifs in the assembly of disulfide-bonded multimers of mucin were also assessed. Cells with plasmids in which both half-cystines in the motif in the D1- or D3-domain of mucin are replaced by alanine expressed proteins that were poorly secreted, suggesting that these mutations impair normal folding of the expressed proteins. A plasmid with a mutant D1-domain motif expressed monomers, whereas one with a mutant D3-domain motif expressed monomers and trimers. However, the trimers expressed by the latter plasmid were assembled in non-acidic compartments, as judged by expression studies in the presence of monensin, which inhibits trimer formation by unmutated plasmid, but not by the mutant plasmid. These results suggest that the CGLCG motif in the D1-domain is required for multimerization in the trans-Golgi complex. However, the CGLCG motif in the D3-domain appears to prevent formation of mucin multimers in non-acidic compartments of the cell. Plasmids encoding the D1- and D2-domains, the D1- and D3-domains, or only the D3-domain also expressed oligomers in the presence of monensin, suggesting that the three D-domains must be contiguous to avoid multimerization in non-acidic compartments. It is possible that these motifs in mucins are engaged in the thiol-disulfide interchange reactions during the assembly of disulfide-bonded multimers of mucin.     

The complete cDNA sequence and structural polymorphism of the polypeptide chain of porcine submaxillary mucin

The complete structure of the DNA encoding the polypeptide chain of porcine submaxillary mucin has been determined. The polypeptide is composed of distinct domains. A large central domain containing tandem repeats of 81 residues each is flanked by much shorter domains with sequences similar to the tandem repeats. Four disulfide-rich domains, three at the amino terminus and one at the carboxyl terminus, complete the chain. The disulfide-rich domains have significant sequence identity to those of other mucins and prepro-von Willebrand factor. The coding region of the mucin gene is highly polymorphic, and three alleles were identified in a single animal that encoded different numbers of the 81-residue tandem repeats. A single large exon devoid of introns encodes the tandem repeat domains. The largest allele with 135 tandem repeats encoded 13,288 amino acids to give a polypeptide with Mr = 1,184,106. The other two alleles contained 99 and 125 tandem repeats, respectively. Each allele also showed different restriction fragment length polymorphisms, which is consistent with the different patterns seen in individual animals. Fragment length polymorphism was also seen within two different families of animals, indicating that the polymorphism observed occurs in a single generation.     

Intrastrand cross-linked actin between Gln-41 and Cys-374. II. Properties of cross-linked oligomers

Actin filaments partially cross-linked with ANP (N-(4-azido-2-nitrophenyl)-putrescine between Gln-41 and Cys-374 on adjacent monomers in the long-pitch helix were depolymerized and fractionated into pools of longitudinal cross-linked dimers (s(o)20,w = 5.55 +/- 0.22 S), trimers (s(o)20,w = 6.93 +/- 0.12 S), and higher-order oligomers. Competition binding experiments of myosin subfragment (S1) to cross-linked dimers in the presence of pyrenyl G-actin revealed about 2 orders of magnitude stronger binding of the first than that of the second S1 molecule to actin dimer. Under similar conditions the unpolymerized cross-linked actin species activated the MgATPase of S1 only severalfold compared to 70-fold activation by F-actin. The cross-linked dimers, trimers, and oligomers were polymerized into filaments by MgCl2 faster than un-cross-linked actin. In electron micrographs these filaments appeared sometimes shorter and had greater tendency to bend than un-cross-linked actin filaments. Small amounts of cross-linked actin dimers nucleated S1-induced polymerization of actin, but the polymerization by S1 was inhibited for pure populations of cross-linked dimers, trimers, and oligomers. The cross-linked dimers did not decrease the kinetic difference between the polymerization of actin by S1 isozymes S1(A1) and S1(A2). According to electron microscopy evidence, cross-linked actin oligomers polymerized by S1 yielded much shorter arrowhead structures than the un-cross-linked actin. These results indicate the importance of lateral actin-actin interaction for the activation of myosin ATPase and the polymerization of actin by S1.     

Porcine submaxillary mucin forms disulfide-linked multimers through its amino-terminal D-domains

COS-7 cells expressing 1,360 residues from the amino terminus of porcine submaxillary mucin were used to determine whether this region, containing the D1, D2, and D3 domains, is involved in forming mucin multimers. Analysis of the proteins immunoprecipitated from the medium of transfected cells by reducing SDS-gel electrophoresis showed a single N-glycosylated protein with no indication of proteolytically processed forms. Without prior reduction, only two proteins, corresponding to monomeric and disulfide-linked trimeric species, were observed. The expressed protein devoid of N-linked oligosaccharides also formed trimers, but was secreted from cells in significantly less amounts than glycosylated trimers. Pulse-chase studies showed that the disulfide-linked trimers were assembled inside the cells no earlier than 30 min after protein synthesis commenced and after the intracellular precursors were N-glycosylated. Trimer formation was inhibited in cells treated with brefeldin A, monensin, chloroquine, or bafilomycin A1, although only brefeldin A prevented the secretion of the protein. These results suggest that trimerization takes place in compartments of the Golgi complex in which the vacuolar H+-ATPase maintains an acidic pH. Coexpression in the same cells of the amino-terminal region and the disulfide-rich carboxyl-terminal domain of the mucin showed that these structures were not disulfide-linked with one another. Cells expressing a DNA construct encoding a fusion protein between the amino- and carboxyl-terminal regions of the mucin secreted disulfide-linked dimeric and high molecular weight multimeric species of the recombinant mucin. The presence of monensin in the medium was without effect on dimerization, but inhibited the formation of disulfide-linked multimers. These studies suggest that disulfide-linked dimers of mucin are subsequently assembled into disulfide-linked multimers by the amino-terminal regions. They also suggest that the porcine mucin forms branched disulfide-linked multimers. This ability of the amino-terminal region of mucin to aid in the assembly of multimers is consistent with its amino acid identities to the amino-terminal region of human von Willebrand factor, which also serves to form disulfide-linked multimers of this protein.     

Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia

A single homozygous nucleotide exchange in exon E3 of the gene encoding the parathyroid hormone receptor type 1 (PTHR1) was identified in an infant with Blomstrand chondrodysplasia born to consanguineous parents. This alteration changes a strictly conserved proline residue at position 132 in the receptor's amino terminal extracellular domain to leucine. COS-1 cells expressing the mutant receptor did not accumulate cyclic adenosine 3',5'-monophosphate in response to PTH or PTH-related peptide (PTHrP) and did not bind the radiolabeled ligand. Expression of the mutant protein on the cell surface of transiently transfected COS-1 cells and in growth plate chondrocytes derived from the affected infant suggests that proline 132 is critical for the receptor's intrinsic binding activity. These findings suggest that the Blomstrand form of human short-limbed dwarfism arises from defective PTHR1 signaling in the developing cartilaginous skeleton.     

Identification of an actin cytoskeletal complex that includes IQGAP and the Cdc42 GTPase

The Rho subfamily of low molecular weight GTPases have been implicated in a variety of cellular functions that include reorganization of the actin cytoskeleton and stress-induced activation of the c-Jun kinase. The downstream targets that mediate the effects of Cdc42 on the actin cytoskeleton have yet to be fully identified. We have used the transient transfection of COS-7 cells with epitope-tagged Cdc42 to identify candidate signaling partners for this GTPase and identified the IQGAP protein as a major in vivo target for activated Cdc42. Epidermal growth factor stimulation of serum-starved COS-7 cells promoted the formation of a Cdc42-IQGAP complex, indicating that growth factors can increase the pool of activated Cdc42. Activated HA-Cdc42 co-localized with IQGAP or F-actin in vivo, whereas cells transfected with dominant-negative forms of Cdc42 (Cdc42(T17N)) showed predominantly dispersed distributions for both HA-Cdc42 and endogenous IQGAP. In detergent lysates from COS-7 cells transiently transfected with different forms of Cdc42, or from stably transfected CHO cells, the induction of actin polymerization by phalloidin resulted in the incorporation of both IQGAP and Cdc42 into actin-containing complexes. Taken together, these findings are consistent with a model whereby IQGAP serves as a target for GTP-bound Cdc42 providing a direct link between the activated GTPase and the actin cytoskeleton.     

Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.     

Chemical cross-linking of a dimeric protein on a modified lectin matrix. A general probe for the chemical topology of oligomeric glycoproteins

A dimeric glycoprotein, glucose oxidase, was allowed to react with lysine-specific cross-linkers, both when immobilized on a succinoylated lectin matrix at a critically low density and also at a high density in solution. Analysis of the cross-linked complexes thus obtained led to the following inferences with regard to the structure of this protein. (1) Of the 15 lysine residues on each glucose oxidase protomer, none is available on the non-interfacial surfaces. (2) Assuming that this protein possesses C2 symmetry with isologous bonding between subunits, it may be inferred that on each promoter there are at least two lysine clusters along or close to the interprotomeric interface. (3) These "interfacial' lysine residues on each protomer are so oriented that the epsilon-amino groups of lysine residues a and b on protomer 1 "face', and are very close to, the epsilon-amino groups of lysine residues b' and a' respectively on protomer 2. General inferences on the geometry of dimeric proteins derivable from an analysis of the cross-linked complexes obtained (as well as those not seen) by using this low-density matrix cross-linking approach were enumerated. Modified lectin matrices may prove useful in studying the three-dimensional structure of glycoproteins, particularly non-crystallizable oligomers.     

Constitutive activation of Mek1 by mutation of serine phosphorylation sites

A variety of extracellular signals lead to the phosphorylation and activation of mitogen-activated protein kinases (MAP kinases). An activator of MAP kinases, Mek1, phosphorylates MAP kinases at threonine and tyrosine residues and is itself phosphorylated at serine-218 and -222 by the protooncogene product Raf-1. By introducing negatively charged residues that may mimic the effect of phosphorylation at positions 218 and 222, we have activated the capacity of Mek1 to phosphorylate MAP kinase by > 100-fold. The most effective activation by a single substitution resulted from the introduction of aspartate at position 218, whereas the introduction of either aspartate or glutamate at position 222 was ineffective. Expression of the activated Mek1 phosphorylation-site mutants in COS-7 cells led to the activation of MAP kinase in the cells and resulted in an increase in the mass of the transfected COS-7 cell population, suggesting an important role of Mek1 in the transduction of mitogenic signals.     

Design, synthesis and utility of novel benzophenone-containing calcitonin analogs for photoaffinity labeling the calcitonin receptor

Calcitonin (CT) is a 32-amino-acid calciotropic peptide hormone which acts on target cells via a G protein-coupled seven-transmembrane receptor (CTR). In this study, we report the design, synthesis and characterization of four potent bioactive and photoreactive CT analogs, each of which contains a single benzophenone moiety inserted at different and discrete locations within the CT molecule. Replacement of all Lys residues in salmon CT (sCT) with Arg, followed by replacement of hydrophobic residues with a Lys(epsilon-p-benzoylbenzoyl) residue [Lys(epsilon-pBz2)] was found to preserve high biological activity. We substituted Val8, Leu16 and Leu19 by Lys(epsilon-pBz2), and acylated the N-terminus by a pBz2 moiety, thus distributing the photoaffinity moiety in the different analogs across a large portion of the CT sequence. With both transfected and endogenous CTRs from several species, all four benzophenone-containing analogs were shown to be virtually indistinguishable from the parent sCT analog in both receptor binding properties and stimulation of cAMP accumulation. Upon photolysis, in the presence of CTR, the radioiodinated photoreactive CT analog ([Arg11,18,Lys19(epsilon-pBz2)]sCT (K19)) covalently labels a membrane component of approximately 70 kDa. Receptor cross-linking is inhibited specifically in the presence of excess sCT. We also examined the interaction of these CT analogs with a hemagglutinin (HA) epitope-tagged CTR. The HA-CTR displayed CT binding and CT-dependent cAMP stimulation identical with native CTR. Both K19 and another bioactive analog (-Arg11,18, Lys8(epsilon-pBz2)]sCT (K8)) specifically photoaffinity cross-link to the HA-CTR. These benzophenone-containing CT analogs should facilitate studies of hormone-receptor interactions and allow the direct identification of a CT binding domain(s) within the receptor by the analysis of photochemically cross-linked conjugates.     

Structural and transglutaminase substrate properties of the small proline-rich 2 family of cornified cell envelope proteins

The small proline-rich (SPR) proteins are components of the cornified cell envelope of stratified squamous epithelia and become cross-linked to other proteins by transglutaminases (TGases). The SPR2 family is the most complex, as it consists of several differentially expressed members of the same size. To explore their physical and cross-linking properties, we have expressed in bacteria a human SPR2 family member, and purified it to homogeneity. By circular dichroism, it possesses no alpha or beta structure but has some organized structure associated with the central peptide repeat domain. The TGase 1, 2, and 3 enzymes expressed in epithelia use the recombinant SPR2 protein as a complete substrate in vitro, but with widely differing kinetic efficiencies, and in different ways. With TGase 1, only one glutamine on the head domain and one lysine on the tail domain were used for limited interchain cross-linking. With TGase 3, multiple head and tail domain residues were used for extensive interchain cross-linking. The total usage of glutamine and lysine residues in vitro by TGase 3 was similar to that seen in earlier in vivo studies. We conclude that SPR2 proteins are cross-linked in epithelia primarily by the TGase 3 enzyme, a minor extent by TGase 1, and probably not by TGase 2.     

N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface

The human Ca2+ receptor (hCaR) is a member of the superfamily of G protein-coupled receptors. Its large (approximately 600 residue) amino-terminal extracellular domain contains 9 potential N-linked glycosylation sites. Immunoblot of cell membranes derived from HEK-293 cells, stably transfected with the hCaR, showed two major immunoreactive bands of approximately 150 and 130 kDa, respectively. Complete digestion of the membranes with PN-glycosidase F yielded a single major immunoreactive band of approximately 115 kDa, confirming the presence of N-linked glycosylation. Treatment of these cells with tunicamycin, which blocks N-linked glycosylation, inhibited signal transduction in response to Ca2+. Flow cytometric analysis showed decreased expression of the hCaR on the cell membrane in tunicamycin-treated cells. Immunoblot of tunicamycin-treated cells showed a reduction in the amount of the 150-kDa band and conversion of the 130-kDa band to the presumptively nonglycosylated 115-kDa form. Tunicamycin treatment of cells, transfected with a mutant hCaR complementary DNA containing a nonsense codon at position 599 preceding the 1st transmembrane domain, blocked the secretion of a 95-kDa protein, representing the amino-terminal extracellular domain, into the medium. These results demonstrate that N-linked glycosylation is required for normal expression of the hCaR at the cell surface.     

Assembly of amino-terminal fibronectin dimers into the extracellular matrix

Fibronectin is a dimeric adhesion molecule that consists of three types of repeating modules. Adherent cells bind soluble fibronectin and incorporate it into insoluble fibrils in the extracellular matrix. The amino-terminal 70-kDa portion of fibronectin mediates binding to the cell surface, but amino-terminal fragments do not accumulate in the extracellular matrix. The ninth type I and first type III modules, the cell adhesion region, and the cysteines that form the interchain disulfide bonds have also been implicated in matrix assembly. To further define which regions of fibronectin are essential for matrix assembly, we generated a dimeric protein (d70 kDa) in which the 70-kDa amino terminus is directly linked to the last 51 amino acids of fibronectin, which contain the cysteines involved in interchain disulfide bonding. d70 kDa bound to cells and accumulated in the extracellular matrix. Incorporation of d70 kDa into the extracellular matrix was dependent upon protein synthesis; in cycloheximide-treated cultures that lacked a pre-existing matrix, d70 kDa accumulated in the extracellular matrix only in the presence of intact fibronectin. Monomeric 70-kDa protein was not incorporated into the matrix in the presence or absence of cycloheximide. These data indicate that fibronectin molecules containing only the amino-terminal 70-kDa region and the carboxyl-terminal 51 amino acids can become assembled into the extracellular matrix.     

Disulfide bridges in the middle part of human fibrinogen

Human fibrinogen contains 29 disulfide bridges per molecule. The amino acid sequences around all half-cystine residues are known. When fibrinogen is cleaved by cyanogen bromide five disulfide-containing fragments are formed. The second-largest of them is derived from the middle part of all three peptide chains, it is monomeric and contains 345 amino acid residues, 12 of which are half-cystines. The arrangement of the six disulfide bonds was determined by analysing sequences and amino acid compositions of subfragments isolated after cleavage with trypsin, thermolysin and staphylococcal protease and after clearage of the disulfide bonds. All half-cystine residues were found to be linked in unique pairs. Six half-cystine residues, two in each of the three peptide chains and forming the -Cys-X-X-X-Cys- sequences, were shown to connect the chains in a ring-like structure, similar to the one in the N-terminal part of the molecule. The remaining six half-cystine residues were found to connect two sections of the gamma-chain in a loop-like structure and four sections of the beta-chain in a loop-inside-a-loop-like structure, the inner beta-chain loop being homologous to the gamma-chain loop.     

The effects of mexiletine, desipramine and fluoxetine in rat models involving central sensitization

The lutropin receptor (LHR) is a G protein-coupled receptor in which high affinity ligand binding occurs to the relatively large extracellular N-terminal domain. Various portions of the receptor have been mapped for their relative importance in localization and in hormone-mediated signaling. There is, however, a paucity of information available on the intracellular loops (ICL), where, along with the C-terminal cytoplasmic tail, G protein coupling is expected to occur. Site-directed mutagenesis was used to investigate the role of several conserved ionizable groups and one tyrosyl residue in ICLs I-III of the rat LHR. The pSVL expression vector, containing the LHR cDNA (wild-type and mutants), was transiently transfected into COS-7 cells, and human choriogonadotropin (hCG) binding and hCG-mediated cAMP production were determined. Several point mutants of amino acid residues in ICL II were prepared and characterized with the following results: replacements of Lys-455 and of His-460 with Glu gave mutant LHRs that failed to localize or fold properly at the cell surface as evidenced by the lack of significant binding to intact cells, although hCG binding could be detected in broken cell preparations, and a neighboring Arg-459 --> Glu replacement had no apparent effect on receptor trafficking, hCG binding or hCG-mediated cAMP-production. A reversal mutant in ICL II in which Glu-441, at the boundary of transmembrane helix III and ICL II, and His-460, at the interface between ICL II and transmembrane helix IV, were interchanged, exhibited hCG binding to intact cells, but the maximal cAMP level at high concentrations of ligand was less than that obtained with COS-7 cells transfected with wild-type LHR. The total number of cell surface receptors determined with the reversal mutant was less than that found with wild-type LHR. This difference, however, is not believed to be responsible for the reduced signaling, since maximal cAMP responses to hCG were obtained with comparable receptor densities of wild-type and various mutant LHRs. Other single replacements in ICL I, Lys-368 --> Glu and to Gln, and in ICL III, Arg-526 --> Glu and Tyr-528 --> Ser, resulted in mutant LHRs with characteristics of wild-type LHR in trafficking, hCG binding and hCG-mediated cAMP production. These findings suggest an important functional role of several amino acid residues in ICL II of LHR.     

Molecular chaperone GRP94 binds to the Fanconi anemia group C protein and regulates its intracellular expression

The FAC protein encoded by the gene defective in Fanconi anemia (FA) complementation group C binds to at least three ubiquitous cytoplasmic proteins in vitro. We used here the complete coding sequence of FAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a B-lymphocyte cDNA library. Binding was confirmed by coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear, lysates of transfected COS-1 cells, as well as from mouse liver cytoplasmic extracts. Deletion mutants of FAC showed that residues 103-308 were required for interaction with GRP94, and a natural splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the cellular phenotype of FA. Our results demonstrate that GRP94 interacts with FAC both in vitro and in vivo and regulates its intracellular level in a cell culture model. In addition, the pathogenicity of the IVS-4 splicing mutation in the FAC gene may be mediated in part by its inability to bind to GRP94.     

Identification of the human ltk gene product in placenta and hematopoietic cell lines

Two different monoclonal antibodies (MAbs) were raised against an extracellular domain and a C-terminal portion of the human ltk protein which is a receptor-type protein tyrosine kinase. Western blot analysis showed that these MAbs specifically immunoprecipitated a 100 kDa ltk protein which was transiently expressed in COS-1 cells transfected with a human ltk cDNA. By an in vitro immune complex kinase assay using these MAbs, a 100 kDa phosphoprotein was detected in human placenta and hematopoietic cell lines. These data indicate that the ltk gene product expressed in human placenta and hematopoietic cells shows tyrosine kinase activity. This is the first detection of native ltk protein naturally expressed in human cells.     

X-linked exudative vitreoretinopathy caused by an arginine to leucine substitution (R121L) in the Norrie disease protein

We report the cosegregation of an arginine to leucine substitution at position 121 of the Norrie disease protein in a large kindred where exudative vitreoretinopathy segregates as an X-linked recessive trait. The clinical phenotype and rate of disease progression were extremely variable, with progression to total retinal detachment from less than age 2 years to more than 21 years. To date, all mutations in X-linked vitreoretinopathy have been missense mutations, presumably not affecting the three-dimensional structure of the NDP gene product, and clustered around residues 121-126 of the Norrie protein. This contrasts with the diversity of mutations seen in the more severe, allelic Norrie disease.     

Alteration of chain length selectivity of a Rhizopus delemar lipase through site-directed mutagenesis

The coding sequences of the Rhizopus delemar lipase and prolipase were altered by oligonucleotide-directed mutagenesis to introduce amino acid substitutions. The resulting mutant enzymes, synthesized by the bacterial host Escherichia coli BL21 (DE3), were tested for their ability to hydrolyze the triglycerides triolein (TO), tricaprylin (TC) and tributyrin (TB). Mutagenesis and lipase gene expression were carried out using plasmid vectors derived from previously described recombinant plasmids [Joerger and Haas (1993) Lipids 28, 81-88] by introduction of the origin of replication of bacteriophage f1. Substitution of threonine 83 (thr83), a residue thought to be involved in oxyanion binding, by alanine essentially eliminated lipolytic activity toward all substrates examined (TB, TO and TC). Replacement of thr83 with serine caused from two- to sevenfold reductions in the activity toward these substrates. Introduction of tryptophan (trp) at position 89, where such a residue is found in closely related fungal lipases, reduced the specific activity toward the three triglyceride substrates. For the mutagenesis of residues in the predicted acyl chain binding groove, mutagenic primers were designed to cause the replacement of a specific codon within the prolipase gene with codons for all other amino acids. Phenylalanine 95 (phe95), phe112, valine 206 (val206) and val209, were targeted. A phenotypic screen was successfully employed to identify cells producing prolipase with altered preference for olive oil, TC or TB. In assays involving equimolar mixtures of the three triglycerides, a prolipase with a phe95-->aspartate mutation showed an almost twofold increase in the relative activity toward TC. Substitution of trp for phe112 caused an almost threefold decrease in the relative preference for TC, but elevated relative TB hydrolysis. Replacement of val209 with trp resulted in an enzyme with a two- and fourfold enhanced preference for TC and TB, respectively.     

Residues in the membrane-spanning and extracellular loop regions of the parathyroid hormone (PTH)-2 receptor determine signaling selectivity for PTH and PTH-related peptide

The parathyroid hormone (PTH)-2 receptor displays strong ligand selectivity in that it responds fully to PTH but not at all to PTH-related peptide (PTHrP). In contrast, the PTH-1 receptor (PTH/PTHrP receptor) responds fully to both ligands. Previously it was shown that two divergent residues in PTH and PTHrP account for PTH-2 receptor selectivity; position 23 (Trp in PTH and Phe in PTHrP) determines binding selectivity and position 5 (Ile in PTH and His in PTHrP) determines signaling selectivity. To identify sites in the PTH-2 receptor involved in discriminating between His5 and Ile5, we constructed PTH-2 receptor/PTH-1 receptor chimeras, expressed them in COS-7 cells, and tested for cAMP responsiveness to [Trp23] PTHrP-(1-36), and to the nondiscriminating peptide [Ile5, Trp23]PTHrP-(1-36) (the Phe23 --> Trp modification enabled high affinity binding of each ligand to the PTH-2 receptor). The chimeras revealed that the membrane-spanning/loop region of the receptor determined His5/Ile5 signaling selectivity. Subsequent analysis of smaller cassette substitutions and then individual point mutations led to the identification of two single residues that function as major determinants of residue 5 signaling selectivity. These residues, Ile244 at the extracellular end of transmembrane helix 3, and Tyr318 at the COOH-terminal portion of extracellular loop 2, are replaced by Leu and Ile in the PTH-1 receptor, respectively. The results thus indicate a functional interaction between two residues in the core region of the PTH-2 receptor and residue 5 of the ligand.