Both G-type domains of protein S are required for the high-affinity interaction with C4b-binding protein.

Anticoagulant protein S interacts with the complement regulatory protein C4b-binding protein (C4BP) via its sex-hormone-binding globulin (SHB6)-like region, which contains two globular (G) domains. Similar G domains are found in Gas6, a protein homologous to protein S, which is not known to bind C4BP or to have any anticoagulant activity. To determine the relative importance of the two G domains in protein S for C4BP protein binding, three recombinant protein S chimeras were produced having either of the two globular domains, or the whole SHB6-like globulin region, replaced by corresponding parts from Gas6. The chimeras were tested for binding to immobilized C4BP using surface-plasmon-resonance technology and microtiter plate-based assays. In both systems, chimeras containing either only globular domains G1 or G2 from protein S were found to bind C4BP. Binding was stimulated by Ca2+ in a manner similar to that found for wild-type protein S. The affinities for C4BP of both chimeras containing individual G domains from protein S, were lower than that of wild-type protein S. Chimera II, containing the G1 domain from protein S, consistently bound C4BP more efficiently than chimera I, which had the protein S-derived G2 domain. The chimera containing the whole SHB6-like globulin region from Gas6 interacted considerably more weakly with C4BP. Our results demonstrate that both G domains of protein S are involved in the interaction between protein S and C4BP and that full affinity binding is dependent on contributions from both domains.     

Functional properties of the sex-hormone-binding globulin (SHBG)-like domain of the anticoagulant protein S.

Protein S (PS) possesses a sex-hormone-binding globulin (SHBG)-like domain in place of the serine-protease domain found in other vitamin K-dependent plasma proteins. This SHBG-like domain is able to bind a complement fraction, C4b-binding protein (C4b-BP). To establish whether the PS SHBG-like domain can fold normally in the absence of other domains, and to obtain information on the specific functions of this region, we expressed the PS SHBG-like domain alone or together with its adjacent domain EGF4. The folding of the two recombinant modules was studied by analyzing their binding to C4b-BP. The apparent dissociation constants of this interaction indicated that both recombinant modules adopted the conformation of native PS, indicating that the PS SHBG-like region is an independent folding unit. We also obtained the first direct evidence that the SHBG-like domain alone is sufficient to support the interaction with C4b-BP. In addition, both recombinant modules were able to bind Ca2+ directly, as shown by the migration shift in agarose gel electrophoresis in the presence of Ca2+, together with the results of equilibrium dialysis and the functional effect of Ca2+ on the C4b-BP/PS interaction, confirming the presence of one Ca2+ binding site within the SHBG-like domain. Neither recombinant module exhibited activated protein C (aPC) cofactor activity in a clotting assay, suggesting that the PS SHBG-like region must be part of the intact molecule for it to contribute to aPC cofactor activity, possibly by constraining the different domains in a conformation that permits optimal interaction with aPC.     

A simple structure encodes G protein-activating function of the IGF-II/mannose 6-phosphate receptor

The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/man6PR) can directly interact with and activate Gi-2, a GTP binding protein (G protein). We found that the segment of residues 2410-2423 in the human IGF-II/man6PR activates Gi-2 in a manner similar to G-coupled receptors. We observed a hierarchy of the segment action when tested on various G proteins, with an order of Gi-2 greater than Gi-1 approximately Gi-3 greater than Go. The segment had no effect on Gs or low molecular weight G proteins. The segment action depended on its primary structure and was potentiated when the segment was connected with a part of the receptor transmembrane region. Finally, the Gi-2-activating function of the human IGF-II/man6PR could be blocked by an antibody against the segment, indicating a critical role for this small region of the receptor.     

The first laminin G-like domain of protein S is essential for binding and activation of Tyro3 receptor and intracellular signalling

The homologous proteins Gas6 and protein S (ProS1) are both natural ligands for the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases. ProS1 selectively activates Tyro3; however, the precise molecular interface of the ProS1-Tyro3 contact has not been characterised. We used a set of chimeric proteins in which each of the C-terminal laminin G-like (LG) domains of ProS1 were swapped with those of Gas6, as well as a set of ProS1 mutants with novel added glycosylations within LG1. Alongside wildtype ProS1, only the chimera containing ProS1 LG1 domain stimulated Tyro3 and Erk phosphorylation in human cancer cells, as determined by Western blot. In contrast, Gas6 and chimeras containing minimally the Gas6 LG1 domain stimulated Axl and Akt phosphorylation. We performed in silico homology modelling and molecular docking analysis to construct and evaluate structural models of both ProS1-Tyro3 and Gas6-Axl ligand-receptor interactions. These analyses revealed a contact between the ProS1 LG1 domain and the first immunoglobulin domain of Tyro3, which was similar to the Gas6-Axl interaction, and involved long-range electrostatic interactions that were further stabilised by hydrophobic and polar contacts. The mutant ProS1 proteins, which had added glycosylations within LG1 but which were all outside of the modelled contact region, all activated Tyro3 in cells with no hindrance. In conclusion, we show that the LG1 domain of ProS1 is necessary for activation of the Tyro3 receptor, involving protein-protein interaction interfaces that are homologous to those of the Gas6-Axl interaction.     

The Ran binding protein RanBPM interacts with Axl and Sky receptor tyrosine kinases

Axl belongs to a particular subfamily of transmembrane receptor tyrosine kinases, the biological ligand for which is the growth/survival factor Gas6. However, little is known about the molecular mechanisms for Axl activation and signal transduction. We have previously identified a novel interaction between the intracellular domain of Axl and Ran binding protein in microtubule organising centre (RanBPM). In the present study, we investigated further the nature of the RanBPM interaction with Axl. A wide distribution of RanBPM mRNA expression in human tissues and various human cancer cell lines was detected. The strength of interaction of both proteins in yeast was comparable to that with the other Axl-binding proteins phosphatidylinositol 3-kinase and Grb2. A truncated version of RanBPM with the SPRY-LisH domain region omitted failed to interact with Axl in yeast. RanBPM was also found to interact in yeast with the Axl homologue, Sky/Tyro3. The interaction between Axl intracellular domain and RanBPM was reproduced in coimmunoprecipitation experiments in both cell-free and mammalian cell systems. Furthermore, coimmunoprecipitation revealed endogenous Axl and RanBPM to interact in several mammalian cell lines in a constitutive manner. Stimulation of COS cells with Gas6 caused increased Axl tyrosine phosphorylation although appeared not to influence the RanBPM-Axl association. In conclusion, we have identified and characterised a novel interaction between RanBPM and the related receptor tyrosine kinases, Axl and Sky. This novel insight into the signalling interactions of Axl and Sky may shed further light on their suspected roles in tumourigenesis, inflammation as well as other cell proliferative diseases.     

Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6

Gas6, a ligand of receptor tyrosine kinases Axl, Sky, and Mer, potentiates cell proliferation and prevents cell death. It also contains g-carboxylglutamic acid residues that mediate the interaction of some blood coagulation factors with negatively charged phospholipids. In our previous study, we demonstrated that Gas6 specifically binds to phosphatidylserine (PS) and links Axl-expressing cells to the PS-coated surface. In this study, to further understand the biological role of the interaction of Gas6 with PS, we examined the effect of Gas6 on the uptake of PS liposomes by macrophages. In vitro phagocytosis studies showed that Gas6 enhanced the uptake of PS liposomes approximately threefold and that the interaction of Gas6 with the surface of macrophages was essential for this enhancement. Analyses of the mechanism of the uptake of PS liposome suggested that Gas6 interacts with PS liposome via its N-terminal Gla domain and with macrophages via its C-terminal domain. Like that of PS liposomes, the uptake of apoptotic cells by macrophages was also enhanced, approximately twofold, in the presence of Gas6. These findings suggest that Gas6 may help phagocytic cells recognize cells with PS exposed on their surfaces, which is considered to be one of the mechanisms for clearing away dying cells. Thus, Gas6 may play a critical role in homeostasis by facilitating the clearance of PS-expressing cells.     

Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces its GDP/GTP exchange rate

The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit.     

Mutations in the carboxy-terminus of the third intracellular loop of the human recombinant VPAC1 receptor impair VIP-stimulated [Ca2+]i increase but not adenylate cyclase stimulation

The vasoactive intestinal polypeptide (VIP) VPAC1 receptor is preferentially coupled to Galphas protein that stimulates adenylate cyclase activity and also to Galphaq and Galphai proteins that stimulate the inositol phosphate/calcium pathway. Previous studies indicated the importance of the third intracellular loop of the receptor for G protein coupling. By site-directed mutation of the human recombinant receptor expressed in Chinese hamster ovary cells, we identified two domains in this loop that contain clusters of basic residues conserved in most of the G-protein-coupled seven transmembrane domains receptors. We found that mutations in the proximal domain (K322) reduced the capability of VIP to increase adenylate cyclase activity without any change in the calcium response, whereas mutations in the distal part of the loop (R338, L339, R341) markedly reduced the calcium increase and Galphai coupling but only weakly the adenylate cyclase activity. Thus, the interaction of different G proteins with the VPAC1 receptor involves different receptor sub-domains.     

Selective interactions between helix VIII of the human mu-opioid receptors and the C terminus of periplakin disrupt G protein activation

Analysis of interactions between the C-terminal tail of the MOP-1 and MOP-1A variants of the human mu-opioid receptor with proteins derived from a human brain cDNA library resulted in identification of the actin and intermediate filament-binding protein periplakin. Mapping of this interaction indicated that the predicted fourth intracellular loop/helix VIII of the receptor interacts with the C-terminal rod and linker region of periplakin. Periplakin is widely expressed in the central nervous system of both man and rat and demonstrated an overlapping but not identical distribution with mu-opioid (MOP) receptors. Co-expression of periplakin with MOP-1 or a MOP-1-eYFP fusion construct in HEK293 cells did not interfere with agonist-mediated internalization of the receptor. When co-expressed with a MOP-1-Gi1 alpha fusion protein periplakin significantly reduced the capacity of the agonist to stimulate binding of 35S-labeled guanosine 5'-3-O-(thio)triphosphate ([35S]GTP gamma S) to the receptor-associated G protein. By contrast, periplakin did not interfere with agonist-stimulation of [35S]GTP gamma S binding to either an alpha 2A-adrenoreceptor-Gi1 alpha fusion protein or a beta2-adrenoreceptor-Gs alpha fusion protein, indicating its selectivity of function. This represents the first example of an opioid receptor-interacting protein that functions to disrupt agonist-mediated G protein activation.     

Two amino acids within the alpha4 helix of Galphai1 mediate coupling with 5-hydroxytryptamine1B receptors.

We previously reported that residues 299-318 in Galphai1 participate in the selective interaction between Galphai1 and the 5-hydroxytryptamine1B (5-HT1B) receptor (Bae, H., Anderson, K., Flood, L. A., Skiba, N. P., Hamm, H. E., and Graber, S. G. (1997) J. Biol. Chem. 272, 32071-32077). The present study more precisely defines which residues within this domain are critical for 5-HT1B receptor-mediated G protein activation. A series of Galphai1/Galphat chimeras and point mutations were reconstituted with Gbetagamma and Sf9 cell membranes containing the 5-HT1B receptor. Functional coupling to 5-HT1B receptors was assessed by 1) [35S]GTPgammaS binding and 2) agonist affinity shift assays. Replacement of the alpha4 helix of Galphai1 (residues 299-308) with the corresponding sequence from Galphat produced a chimera (Chi22) that only weakly coupled to the 5-HT1B receptor. In contrast, substitution of residues within the alpha4-beta6 loop region of Galphai1 (residues 309-318) with the corresponding sequence in Galphat either permitted full 5-HT1B receptor coupling to the chimera (Chi24) or only minimally reduced coupling to the chimeric protein (Chi25). Two mutations within the alpha4 helix of Galphai1 (Q304K and E308L) reduced agonist-stimulated [35S]GTPgammaS binding, and the effects of these mutations were additive. The opposite substitutions within Chi22 (K300Q and L304E) restored 5-HT1B receptor coupling, and again the effects of the two mutations were additive. Mutations of other residues within the alpha4 helix of Galphai1 had minimal to no effect on 5-HT1B coupling behavior. These data provide evidence that alpha4 helix residues in Galphai participate in directing specific receptor interactions and suggest that Gln304 and Glu308 of Galphai1 act in concert to mediate the ability of the 5-HT1B receptor to couple specifically to inhibitory G proteins.     

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.     

Three-dimensional model of the SHBG-like region of anticoagulant protein S: new structure-function insights

Protein S (PS) is a vitamin K-dependent glycoprotein that consists of several modules including a C-terminal sex hormone-binding globulin (SHBG)-like domain that has been subdivided into two laminin LG-type domains. The SHBG-like region of PS is known to bind to a complement regulator molecule, C4b-binding protein (C4BP), coagulation factor Va (FVa) and receptor tyrosine kinases. Inherited PS deficiency has been associated with thromboembolic disease. Yet, study of the mechanisms by which the SHBG-like region of PS serves its essential functions has so far been hampered because of the lack of structural information. Recently, the three-dimensional (3D) structure of LG domains from plasma SHBG, laminin and neurexin have been reported and were found related to the pentraxin family. We used these X-ray structures to build homology models of the SHBG-like region of human PS. We then analyzed previously reported experimental/clinical data in the light of the predicted structures. A potential calcium-binding site is found in the first LG domain of PS and D292 could play a role in this process. This region is close to the interface between the two LG domains and is also surrounded by segments that have been suggested by synthetic peptide studies to be important for C4BP or FVa binding. The 39 point mutations linked to PS deficiencies or reported as neutral variants were rationalized in the 3D structure. Proteins 2001;43:203-216.     

Prediction of solution structures of the Ca2+-bound gamma-carboxyglutamic acid domains of protein S and homolog growth arrest specific protein 6: use of the particle mesh Ewald method.

The solution structures of the N-terminal domains of protein S, a plasma vitamin K-dependent glycoprotein, and its homolog growth arrest specific protein 6 (Gas6) were predicted by molecular dynamics computer simulations. The initial structures were based on the x-ray crystallographic structure of the corresponding region of bovine prothrombin fragment 1. The subsequent molecular dynamics trajectories were calculated using the second-generation AMBER force field. The long-range electrostatic forces were evaluated by the particle mesh Ewald method. The structures that stabilized over a 400-ps time interval were compared with the corresponding region of the simulated solution structure of bovine prothrombin fragment 1. Structural properties of the gamma-carboxyglutamic acid (Gla) domains obtained from simulations and calcium binding were found to be conserved for all three proteins. Analysis of the predicted solution structure of the Gla domain of Gas6 suggests that this domain should bind with negatively charged phospholipid surfaces analogous to bovine prothrombin fragment 1 and protein S.     

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the alpha subunit of GS protein

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.     

Molecular mechanism of the vitamin D antagonistic actions of (23S)-25-dehydro-1alpha-hydroxyvitamin D3-26,23-lactone depends on the primary structure of the carboxyl-terminal region of the vitamin d receptor

We reported that (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) antagonizes vitamin D receptor (VDR)-mediated genomic actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] in human cells but is agonistic in rodent cells. Human and rat VDR ligand-binding domains are similar, but differences in the C-terminal region are important for ligand binding and transactivation and might determine the agonistic/antagonistic effects of TEI-9647. We tested TEI-9647 on 1alpha,25(OH)(2)D(3) transactivation using SaOS-2 cells (human osteosarcoma) or ROS 24/1 cells (rat osteosarcoma) cotransfected with human or rodent VDR and a reporter. In both cell lines, TEI-9647 was antagonistic with wild-type human (h)VDR, but agonistic with overexpressed wild-type rat (r)VDR. VDR chimeras substituting the hVDR C-terminal region (activation function 2 domain) with corresponding rVDR residues diminished antagonism and increased agonism of TEI-9647. However, substitution of 25 C-terminal rVDR residues with corresponding hVDR residues diminished agonism and increased antagonism of TEI-9647. hVDR mutants (C403S, C410N) demonstrated that Cys403 and/or 410 was necessary for TEI-9647 antagonism of 1alpha,25(OH)(2)D(3) transactivation. These results suggest that species specificity of VDR, especially in the C-terminal region, determines the agonistic/antagonistic effects of TEI-9647 that determine, in part, VDR interactions with coactivators and emphasize the critical interaction between TEI-9647 and the two C-terminal hVDR Cys residues to mediate the antagonistic effect of TEI-9647.     

Insulin-like growth factor-II/mannose 6-phosphate receptor is incapable of activating GTP-binding proteins in response to mannose 6-phosphate, but capable in response to insulin-like growth factor-II

We previously reported that insulin-like growth factor-II (IGF-II) stimulates both calcium influx and DNA synthesis by acting on the cell surface IGF-II receptor (IGF-IIR) in a manner sensitive to pertussis toxin, and recently demonstrated that IGF-II binding to the IGF-IIR gives rise to functional changes of purified Gi-2, a GTP-binding protein (G protein) in phospholipid vesicles as well as in broken cell membranes. On the other hand, a variety of evidence indicates that the IGF-IIR binds mannose 6-phosphate (man6P) with high affinity probably at a receptor extracellular region different from the IGF-II-binding site. In the present study, we examined whether man6P stimulation of the IGF-IIR evokes the activation of Gi-2 in phospholipid vesicles and in native cell membranes. In vesicles reconstituted with purified rat IGF-IIR and bovine Gi-2, man6P did not stimulate GDP dissociation from Gi-2 even in concentrations up to 10 mM, while IGF-II dose-dependently facilitated GDP release from Gi-2 with an EC50 of 6 nM. The stimulatory effect of IGF-II was not observed in vesicles reconstituted with Gi-2 alone. In addition, also in a native environment of cell membranes, man6P did not affect an endogenous 40-kDa protein or exogenously added purified Gi-2 as assessed with reduction of the pertussis toxin-catalyzed ADP-ribosylation. These results indicate that the IGF-IIR does not activate Gi-like proteins upon man6P binding in phospholipid vesicles and in native cellular membranes, whereas the receptor activates Gi-like proteins upon IGF-II binding in both environments. Thus, we postulate that the IGF-IIR dissimilarly responds to the two structurally unrelated ligands, IGF-II and man6P, in the linkage function with G proteins.     

Identification of effector-activating residues of Gs alpha.

The heterotrimeric G proteins transduce extracellular signals by interacting with specific intracellular effectors. We have used a scanning mutagenesis approach to identify amino acids of alpha S, the alpha subunit of Gs, that determine the specificity of its interaction with its effector, adenylyl cyclase. In alpha subunit chimeras, residues 236-356 of alpha S comprise the shortest linear stretch that is required for activation of adenylyl cyclase. Within these 121 residues, we identified four clusters of residues in which substitutions prevented effector activation. Mutations in three of these regions did not affect alpha subunit expression or the GTP-induced conformational change. The identified alpha S residues in the NH2-terminal half of the 121-residue region endowed the cognate alpha i2 segment with the ability to activate effector, while those in the COOH-terminal half did not. In a three-dimensional G alpha model, based on the structure of p21ras, the effector-activating residues of alpha S form a surface on the membrane-facing side of the molecule; this surface includes a region that changes conformation upon binding GTP.     

Influence of the Hinge Region and Its Adjacent Domains on Binding and Signaling Patterns of the Thyrotropin and Follitropin Receptor

Glycoprotein hormone receptors (GPHR) have a large extracellular domain (ECD) divided into the leucine rich repeat (LRR) domain for binding of the glycoprotein hormones and the hinge region (HinR), which connects the LRR domain with the transmembrane domain (TMD). Understanding of the activation mechanism of GPHRs is hindered by the unknown interaction of the ECD with the TMD and the structural changes upon ligand binding responsible for receptor activation. Recently, our group showed that the HinR of the thyrotropin receptor (TSHR) can be replaced by those of the follitropin (FSHR) and lutropin receptor (LHCGR) without effects on surface expression and hTSH signaling. However, differences in binding characteristics for bovine TSH at the various HinRs were obvious. To gain further insights into the interplay between LRR domain, HinR and TMD we generated chimeras between the TSHR and FSHR. Our results obtained by the determination of cell surface expression, ligand binding and G protein activation confirm the similar characteristics of GPHR HinRs but they also demonstrate an involvement of the HinR in ligand selectivity indicated by the observed promiscuity of some chimeras. While the TSHR HinR contributes to specific binding of TSH and its variants, no such contribution is observed for FSH and its analog TR4401 at the HinR of the FSHR. Furthermore, the charge distribution at the poorly characterized LRR domain/HinR transition affected ligand binding and signaling even though this area is not in direct contact with the ligand. In addition our results also demonstrate the importance of the TMD/HinR interface. Especially the combination of the TSHR HinR with the FSHR-TMD resulted in a loss of cell surface expression of the respective chimeras. In conclusion, the HinRs of GPHRs do not only share similar characteristics but also behave as ligand specific structural and functional entities.     

Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.