Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Evidence for a novel DNA damage binding protein in human cells.

We describe a novel DNA damage binding activity in nuclear extracts from a normal human fibroblast cell strain. This protein was identified using electrophoretic mobility shift assays of immunopurified UV-irradiated oligonucleotide substrates containing a single, site-specific cyclobutane pyrimidine dimer or a pyrimidine (6-4) pyrimidinone photoproduct. Compared with the (6-4) photoproduct, which displayed similar levels of binding in double and single-stranded substrates, the protein showed somewhat lower affinity for the cyclobutane dimer in a single-stranded oligonucleotide and negligible binding in double-stranded DNA. The specificity and magnitude of binding was similar in cells with normal excision repair (GM637) and repair-deficient cells from xeroderma pigmentosum groups A (XP12RO) and E (XP2RO). An apparent molecular mass of 66 kDa consisting of two subunits of approximately 22 and approximately 44 kDa was determined by Southwestern analysis. Cell cycle studies using centrifugal cell elutriation indicated that the binding activity was significantly greater in G1 phase compared with S phase in a human lymphoblast cell line. Gel supershift analysis using an anti-replication protein A antibody showed that the binding protein was not antigenically related to the human single-stranded binding protein. Taken together, these data suggest that this activity represents a novel DNA damage binding protein that, in addition to a putative role in excision repair, may also function in cell cycle or gene regulation.

ATP-dependent uptake of natural product cytotoxic drugs by membrane vesicles establishes MRP as a broad specificity transporter.

MRP is a recently isolated ATP-binding cassette family transporter. We previously reported transfection studies that established that MRP confers multidrug resistance [Kruh, G. D., Chan, A., Myers, K., Gaughan, K., Miki, T. & Aaronson, S. A. (1994) Cancer Res. 54, 1649-1652] and that expression of MRP is associated with enhanced cellular efflux of lipophilic cytotoxic agents [Breuninger, L. M., Paul, S., Gaughan, K., Miki, T., Chan, A., Aaronson, S. A. & Kruh, G. D. (1995) Cancer Res. 55, 5342-5347]. To examine the biochemical mechanism by which MRP confers multidrug resistance, drug uptake experiments were performed using inside-out membrane vesicles prepared from NIH 3T3 cells transfected with an MRP expression vector. ATP-dependent transport was observed for several lipophilic cytotoxic agents including daunorubicin, etoposide, and vincristine, as well as for the glutathione conjugate leukotriene C4 (LTC4). However, only marginally increased uptake was observed for vinblastine and Taxol. Drug uptake was osmotically sensitive and saturable with regard to substrate concentration, with Km values of 6.3 microM, 4.4 microM, 4.2 microM, 35 nM, and 38 microM, for daunorubicin, etoposide, vincristine, LTC4, and ATP, respectively. The broad substrate specificity of MRP was confirmed by the observation that daunorubicin transport was competitively inhibited by reduced and oxidized glutathione, the glutathione conjugates S-(p-azidophenacyl)-glutathione (APA-SG) and S-(2,4-dinitrophenyl)glutathione (DNP-SG), arsenate, and the LTD4 antagonist MK571. This study establishes that MRP pumps unaltered lipophilic cytotoxic drugs, and suggests that this activity is an important mechanism by which the transporter confers multidrug resistance. The present study also indicates that the substrate specificity of MRP is overlapping but distinct from that of P-glycoprotein, and includes both the neutral or mildly cationic natural product cytotoxic drugs and the anionic products of glutathione conjugation. The widespread expression of MRP in tissues, combined with its ability to transport both lipophilic xenobiotics and the products of phase II detoxification, indicates that the transporter represents a widespread and remarkably versatile cellular defense mechanism.

Binding site size limit of the 2:1 pyrrole-imidazole polyamide-DNA motif.

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids can be combined in antiparallel side-by-side dimeric complexes for sequence-specific recognition in the minor groove of DNA. Six polyamides containing three to eight rings bind DNA sites 5-10 bp in length, respectively. Quantitative DNase I footprint titration experiments demonstrate that affinity maximizes and is similar at ring sizes of five, six, and seven. Sequence specificity decreases as the length of the polyamides increases beyond five rings. These results provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity.

Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS.

The MADS domain homeotic proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) act in a combinatorial manner to specify the identity of Arabidopsis floral organs. The molecular basis for this combinatorial mode of action was investigated. Immunoprecipitation experiments indicate that all four proteins are capable of interacting with each other. However, these proteins exhibit "partner-specificity" for the formation of DNA-binding dimers; only AP1 homodimers, AG homodimers, and AP3/PI heterodimers are capable of binding to CArG-box sequences. Both the AP3/PI heterodimer and the AP1 or AG homodimers are formed when the three corresponding proteins are present together. The use of chimeric proteins formed by domain swapping indicates that the L region (which follows the MADS box) constitutes a key molecular determinant for the selective formation of DNA-binding dimers. The implications of these results for the ABC genetic model of flower development are discussed.

A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in the brain.

The Shc adaptor protein, hereafter referred to as ShcA, possesses two distinct phosphotyrosine-recognition modules, a C-terminal Src homology 2 (SH2) domain and an N-terminal phosphotyrosine-binding (PTB) domain, and is itself phosphorylated on tyrosine in response to many extracellular signals. Phosphorylation of human ShcA at Tyr-317 within its central (CH1) region induces binding to the Grb2 SH2 domain and is thereby implicated in activation of the Ras pathway. Two shc-related genes (shcB and shcC) have been identified in the mouse. shcB is closely related to human SCK, while shcC has not yet been found in other organisms. The ShcC protein is predicted to have a C-terminal SH2 domain, a CH1 region with a putative Grb2-binding site, and an N-terminal PTB domain. The ShcC and ShcB SH2 domains bind phosphotyrosine-containing peptides and receptors with a specificity related to, but distinct from, that of the ShcA SH2 domain. The ShcC PTB domain specifically associates in vitro with the autophosphorylated receptors for nerve growth factor and epidermal growth factor. These results indicate that ShcC has functional SH2 and PTB; domains. In contrast to shcA, which is widely expressed, shcC RNA and proteins are predominantly expressed in the adult brain. These results suggest that ShcC may mediate signaling from tyrosine kinases in the nervous system, such as receptors for neurotrophins.

Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase.

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl] -4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33 angstroms resolution and refined to an Rfactor 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2.

Batimastat, a potent matrix mealloproteinase inhibitor, exhibits an unexpected mode of binding.

Matrix metalloproteinase enzymes have been implicated in degenerative processes like tumor cell invasion, metastasis, and arthritis. Specific metalloproteinase inhibitors have been used to block tumor cell proliferation. We have examined the interaction of batimastat (BB-94) with a metalloproteinase [atrolysin C (Ht-d), EC 3.4.24.42] active site at 2.0-angstroms resolution (R = 16.8%). The title structure exhibits an unexpected binding geometry, with the thiophene ring deeply inserted into the primary specificity site. This unprecedented binding geometry dramatizes the significance of the cavernous primary specificity site, pointing the way for the design of a new generation of potential antitumor drugs.

Sequence specificity of triplex DNA formation: Analysis by a combinatorial approach, restriction endonuclease protection selection and amplification.

We have devised a combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identify consensus ligand binding sequences in DNA. In this technique, cleavage by a type IIS restriction endonuclease (an enzyme that cleaves DNA at a site distal from its recognition sequence) is prevented by a bound ligand while unbound DNA is cleaved. Since the selection step of REPSA is performed in solution under mild conditions, this approach is amenable to the investigation of ligand-DNA complexes that are either insufficiently stable or not readily separable by other methods. Here we report the use of REPSA to identify the consensus duplex DNA sequence recognized by a G/T-rich oligodeoxyribonucleotide under conditions favoring purine-motif triple-helix formation. Analysis of 47 sequences indicated that recognition between 13 bases on the oligonucleotide 3' end and the duplex DNA was sufficient for triplex formation and indicated the possible existence of a new base triplet, G.AT. This information should help identify appropriate target sequences for purine-motif triplex formation and demonstrates the power of REPSA for investigating ligand-DNA interactions.

In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities.

A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'-A5T4T3G2C1G1'C2'A3A4'T5'-3'. Mutants containing the sequence Leu-18Tyr-15Xaa-14Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed.

Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A+T-rich elements.

The Drosophila CF2II protein, which contains zinc fingers of the Cys2His2 type and recognizes an A+T-rich sequence, behaves in cell culture as an activator of a reporter chloramphenicol acetyltransferase gene. This activity depends on C-terminal but not N-terminal zinc fingers, as does in vitro DNA binding. By site-specific mutagenesis and binding site selection, we define the critical amino acid-base interactions. Mutations of single amino acid residues at the leading edge of the recognition helix are rarely neutral: many result in a slight change in affinity for the ideal DNA target site; some cause major loss of affinity; and others change specificity for as many as two bases in the target site. Compared to zinc fingers that recognize G+C-rich DNA, CF2II fingers appear to bind to A+T-rich DNA in a generally similar manner, but with additional flexibility and amino acid-base interactions. The results illustrate how zinc fingers may be evolving to recognize an unusually diverse set of DNA sequences.

Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites.

The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding. The Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.

A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity.

The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of kcat/Km). Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD. The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NAD-dependent enzyme. Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences--strict adherence to homology as a criterion for replacing these amino acids impairs function. Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes.

Major histocompatibility complex binding affinity of an antigenic determinant is crucial for the differential secretion of interleukin 4/5 or interferon gamma by T cells.

Differential activation of CD4+ T-cell precursors in vivo leads to the development of effectors with unique patterns of lymphokine secretion. To investigate whether the differential pattern of lymphokine secretion is influenced by factors associated with either the display and/or recognition of the ligand, we have used a set of ligands with various class II binding affinities but unchanged T-cell specificity. The ligand that exhibited approximately 10,000-fold higher binding to I-Au considerably increased the frequency of interferon gamma-producing but not interleukin (IL) 4- or IL-5-secreting cells in vivo. Using an established ligand-specific, CD4+ T-cell clone secreting only IL-4, we also demonstrated that stimulation with the highest affinity ligand resulted in interferon gamma production in vitro. In contrast, ligands that demonstrated relatively lower class II binding induced only IL-4 secretion. These data suggest that the major histocompatibility complex binding affinity of antigenic determinants, leading to differential interactions at the T cell-antigen-presenting cell interface, can be crucial for the differential development of cytokine patterns in T cells.