Effective partnering of academic and physician scientists with the pharmaceutical drug development industry.

This manuscript briefly addresses the drug discovery and development process. It is a long road from the formulation of a good discovery idea to the acceptance of a new drug in the marketplace, and there are many challenges faced along the way to the patient. Collaborations and partnerships are an important part of this process. There are a variety of partnering opportunities, ranging from the discovery of novel technologies and drug targets to lead discovery, compound gifts, and external sourcing. These partnerships help increase confidence and improve decision making on issues of safety and efficacy preclinically, which can reduce attrition and expedite the provision of new quality drugs to patients more quickly and at lower costs. Collaborations involve addressing multiple issues that include infrastructure, safety, regulatory matters, intellectual property, technical and personnel considerations, source document capture and data analysis issues, and legal and strategic alliances. A number of success factors are identified as important for quality collaborations in the drug development process.

Cutting edge: a small molecule antagonist of LFA-1-mediated cell adhesion.

LFA-1 (CD18,CD11a) is a cell-adhesion molecule that mediates critical immunological processes. In this paper we report the discovery and characterization of (R)-5-(4-bromobenzyl)-3-(3, 5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione (BIRT 377), an orally bioavailable small molecule that interacts specifically with LFA-1 via noncovalent binding to the CD11a chain and prevents LFA-1 from binding to its ligand, ICAM-1. BIRT 377 inhibits lymphocyte activity both in vitro and in vivo, in functional assays that require LFA-1-mediated cell adhesion. These results demonstrate that LFA-1-mediated leukocyte adhesion can be antagonized with noncharged, low m.w. molecules and suggest that the potential therapeutic value of adhesion inhibitors can be attained with a small, orally bioavailable compound.

Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues, and cells.

The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.

Molecular comparison of soluble intercellular adhesion molecule (sICAM)-1 and sICAM-3 binding to lymphocyte function-associated antigen-1.

The interactions of intercellular adhesion molecules-1 and -3 (ICAM-1 and ICAM-3) with lymphocyte function-associated antigen-1 (LFA-1) have been characterized and compared on the molecular and cellular level. Enzyme-linked immunosorbent-based molecular assays have been utilized to calculate the binding affinities of soluble ICAM-1 (sICAM-1) and soluble ICAM-3 (sICAM-3) for LFA-1. Consistent with previously published data, we found that sICAM-1 binds to LFA-1 with an affinity of approximately 60 nM. In contrast, sICAM-3 binds to LFA-1 with an affinity approximately 9 times weaker ( approximately 550 nM). Both sICAM-1 and sICAM-3 require divalent cations for binding. Specifically, both Mg2+ and Mn2+ support high affinity adhesion, although interestingly, high concentrations of Ca2+ decrease the affinity of each molecule for LFA-1 substantially. Furthermore, a panel of anti-LFA-1 monoclonal antibodies were characterized for their ability to block sICAM-1 and sICAM-3/LFA-1 interactions in molecular and cellular assays to help distinguish binding sites on LFA-1 for both molecules. Finally, molecular and cellular competition experiments demonstrate that sICAM-1 and sICAM-3 compete with each other for binding to LFA-1. The above data demonstrate that sICAM-1 and sICAM-3 share a common binding site or an overlapping binding site on LFA-1 and that the apparent differences in binding sites can be attributed to different affinities of sICAM-1 and sICAM-3 for LFA-1.

Effect of deoxycoformycin and Val-boroPro on the associated catalytic activities of lymphocyte CD26 and ecto-adenosine deaminase.

CD26 and ecto-adenosine deaminase (ADA) are found associated on the plasma membrane of T lymphocytes and each possess distinct catalytic activities. CD26 has a proteolytic activity identical to dipeptidylpeptidase IV (DPPIV; E.C. 3.4.14.5), and ecto-ADA (E.C. 3.5.4.4) degrades extracellular adenosine. The cell surface expression of CD26 and ecto-adenosine deaminase (ecto-ADA) is regulated on stimulated T lymphocytes, and ADA binding to CD26 produces a synergistic costimulatory response with T cell receptor activation. This study addresses the potential regulation by allosteric interactions of the catalytic activities of CD26 associated with ecto-ADA, which could define the mechanism of the synergism observed in T cell signaling. Cell lines genetically deficient in ADA, ligands for ADA such as adenosine, and a specific inhibitor of ADA, deoxycoformycin, were used to define the effect of ADA activity on CD26 DPPIV activity and affinity for dipeptide substrate. Conversely, a recombinant Chinese hamster ovary cell line expressing human CD26 with or without a mutation in the DPPIV catalytic domain, and the boronic acid inhibitor Val-boroPro, were used to determine the effect of DPPIV activity on ecto-ADA activity and association with CD26. These studies found no significant allosteric interaction between the catalytic activities of CD26 and ecto-ADA when associated. Therefore, signaling events in T cells involving costimulation with CD26 and ecto-ADA and the synergism observed upon ADA binding to CD26 occur independently of the catalytic activities of these cell surface molecules.

Characterization of molecular interactions between intercellular adhesion molecule-1 and leukocyte function- associated antigen-1.

The ability of soluble ICAM-1 (sICAM-1) to inhibit ICAM-1/LFA-1 adhesion events has been reported previously by numerous investigators. sICAM-1 has been demonstrated to inhibit various in vitro assays at concentrations ranging from 2 nM to greater than 40 microM. Given the hypothesis that circulating ICAM-1 modulates immune functions, the ability of sICAM-1 to inhibit cellular functions may have significant ramifications. Considering the potential clinical importance of the interaction between ICAM-1 and its receptor, LFA-1, it is necessary to understand this receptor-ligand interaction at a molecular level. In this study, direct binding experiments were utilized to determine the affinity between biotinylated monomeric sICAM-1 and immobilized LFA-1 (approximately 130 nM). Competitive binding experiments with unlabeled sICAM-1 and a truncated form of sICAM-1 (D1D2) yielded similar affinities. The specificity of this interaction was characterized using mAbs directed against sICAM-1 or LFA-1. This assay system was extended to include multimeric species using nonblocking mAbs directed against domains D4 and D5 of sICAM-1. Dimerizing sICAM-1 with a mAb alphaD4 or alphaD5 increased the affinity for immobilized LFA-1 by two orders of magnitude (approximately 4 nM), an effect presumably due to avidity. These results indicate that while the monomeric sICAM-1/LFA-1 interaction may involve only a moderate binding affinity, multimeric ICAM-1 present on a cell surface may bind cell surface-immobilized LFA-1 with very high avidity. These sICAM-1/LFA-1 molecular assays should be useful in defining the efficacy of potential antagonists.

Strong hydrogen bonding interactions involving a buried glutamic acid in the transmembrane sequence of the neu/erbB-2 receptor.

The receptor tyrosine kinase encoded by the neu/erbB-2 proto-oncogene is constitutively activated by a single valine to glutamic acid substitution at position 664 in the predicted membrane-spanning sequence of the receptor. We have explored the structural changes involved in receptor activation with polarized FTIR and magic angle spinning NMR spectroscopy. The hydrophobic transmembrane sequence folds into a well-defined alpha-helical structure spanning the membrane bilayer. Measurements of the pKa and 13C chemical shift anisotropy of Glu 664 reveal that the side chain carboxyl group is protonated and strongly hydrogen bonded. These studies provide direct evidence for glutamate hydrogen-bonding interactions in the mechanism of receptor dimerization and activation.

The native structure of intercellular adhesion molecule-1 (ICAM-1) is a dimer. Correlation with binding to LFA-1.

In solution, intercellular adhesion molecule-1 (ICAM-1) exhibits extremely low affinity for its receptor, LFA-1, as direct binding to LFA-1 has not been reported. Furthermore, there are conflicting reports on the ability of ICAM-1 in solution to inhibit cell adhesion events. These differences could be due to the valency or an oligomeric native biochemical form of membrane-bound and soluble ICAM-1, which may correlate with its ability to bind to integrins. To test this, stimulated adenocarcinoma (A549) cells or HUVEC were labeled with 35S-methionine/cysteine and treated with a chemical cross-linker. A high m.w. form (200 kDa) of ICAM-1 but not ICAM-2 was specifically immunoprecipitated from cross-linked cell lysates and supernatants. Affinity purification of crosslinked supernatants revealed that the majority of ICAM-1 was dimeric as opposed to recombinant soluble ICAM-1, which contains a minor fraction of dimer. Gel filtration chromatography was used to isolate monomeric and dimer-rich fractions of recombinant soluble ICAM-1, and tested for direct binding to affinity-purified LFA-1. Dimer-rich fractions demonstrated an enhanced ability and estimated affinity, compared with monomeric protein, to bind to purified LFA-1. These data suggest that ICAM-1 exists in its native membrane-bound and shed form as a non-covalent dimer, and that dimerization directly correlates with enhanced binding to LFA-1.

Determination of helix-helix interactions in membranes by rotational resonance NMR.

Dimerization of human glycophorin A in erythrocyte membranes is mediated by specific interactions within the helical transmembrane domain of the protein. Rotational resonance NMR provides a unique approach for obtaining high-resolution structural data in membrane systems and has been used to establish intermolecular contacts in the glycophorin A dimer by using hydrophobic peptides that correspond to the transmembrane sequence. Magnetization exchange rates were measured between [13C]methyl labels in the hydrophobic sequence -G79-V80-M81-A82-G83-V84- located in the middle of the transmembrane domain and specific [13C]carbonyl labels along the peptide backbone across the dimer interface. Significant magnetization exchange was observed only between V80 (13CH3) and G79 (13C = O) and between V84 (13CH3) and G83 (13C = O), indicating that these residues are packed in the dimer interface in a "ridges-ingrooves" arrangement.

Structure and orientation of the transmembrane domain of glycophorin A in lipid bilayers.

Rotational resonance (RR) NMR, circular dichroism (CD), and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy are used to establish the secondary structure and orientation of peptides corresponding to the transmembrane domain of human glycophorin A in dimyristoylphosphatidylcholine bilayers. An amide I vibrational frequency of 1650 cm-1 and negative CD absorption bands at 208 and 222 nm indicate that the peptide is largely alpha-helical, while an order parameter of 0.35-0.50 in the ATR-FTIR measurements indicates that the peptide orientation is generally perpendicular to the bilayer plane. High-resolution structural data on the glycophorin A transmembrane (GPA-TM) peptides were obtained by measuring the rate of magnetization exchange between pairs of specific 13C labels using RR NMR. The exchange rates are translated into internuclear distances with a resolution on the order of 0.3 A. These experiments are similar in design to previous experiments on crystalline peptides where the 13C labels were incorporated into amino acids separated by 2-3 residues in the peptide sequence but close together in space due to a helical peptide geometry [Peersen, O.B., Yoshimura, S., Hojo, H., Aimoto, S., & Smith, S.O. (1992) J. Am. Chem. Soc. 114, 4332-4335]. In the GPA-TM peptides, magnetization exchange rates measured between [1-13C]V80 and [2-13C] G83 between [1-13C]M81 and [2-13C]G83 in the middle of the transmembrane sequence correspond to internuclear distances of approximately 4.5 A and are consistent with a helical peptide structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Rotational resonance NMR determination of intra- and intermolecular distance constraints in dipalmitoylphosphatidylcholine bilayers.

Rotational resonance (RR) NMR methods are explored for determining intramolecular and intermolecular distances between 13C-labeled sites in membrane bilayers. Specific 13C labels have been incorporated into dipalmitoylphosphatidylcholine (DPPC) and a hydrophobic peptide corresponding to the transmembrane domain of human glycophorin A. The exchange of magnetization between these labels in the RR NMR experiment can be related to their internuclear separation. Intramolecular magnetization exchange rates have been measured between 13C labels incorporated at the 1-position of the sn-1 acyl chain (1-[1-13C]) and the 2-position of the sn-2 acyl chain (2-[2-13C]) of DPPC. These positions are 5.3-5.5 A apart in the crystal structure of dimyristoylphosphatidylcholine (DMPC), but are estimated to be 4.5-5.0 A apart in DPPC below the subphase transition temperature. These results are consistent with a smaller axial displacement between the sn-1 and sn-2 acyl chains than the approximately 3.6-A displacement observed in the DMPC crystal structure. Intermolecular magnetization transfer rates have been measured between 1,2-[2-13C]DPPC and 2-[1,13C]DPPC and between 1,2-[1-13C]DPPC and 2-[2-13C]DPPC. In addition, intermolecular magnetization exchange rates have been measured between 1,2-[2-13C]DPPC and the 13C-OH position of Y93 in the glycophorin transmembrane domain. These intermolecular distance measurements demonstrate that the relative orientation and location of membrane lipids and peptides can be established using RR NMR methods.

An extra cysteine proximal to the transmembrane domain induces differential cross-linking of p185neu and p185neu.

The neu proto-oncogene encodes a receptor tyrosine kinase (p185) that is closely related to the epidermal growth factor receptor. It has been proposed that receptor tyrosine kinases are activated through oligomerization. Because this clustering model predicts that oligomerization of receptors is sufficient to activate them, we determined if p185 can be activated by introducing an extra cysteine proximal to the transmembrane domain. This should induce inter-receptor disulfide bonding and, according to the clustering model, activate the receptor. This amino acid substitution enhanced recovery of both normal and transforming neu proteins as dimers, with normal p185 recovered predominantly as monomers and transforming p185* as dimers. However, the cysteine substitution did not affect the transforming activity of the two proteins.

Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices.

Specific side-by-side interactions between transmembrane alpha-helices may be important in the assembly and function of integral membrane proteins. We describe a system for the genetic and biophysical analysis of these interactions. The transmembrane alpha-helical domain of interest is fused to the C-terminus of staphylococcal nuclease. The resulting chimera can be expressed at high levels in Escherichia coli and is readily purified. In our initial application we study the single transmembrane alpha-helix of human glycophorin A (GpA), thought to mediate the SDS-stable dimerization of this protein. The resulting chimera forms a dimer in SDS, which is disrupted upon addition of a peptide corresponding to the transmembrane domain of GpA. Deletion mutagenesis has been used to delineate the minimum transmembrane domain sufficient for this behavior. Site-specific mutagenesis shows that a methionine residue, previously implicated as a potential interfacial residue, can be replaced with other hydrophobic residues without disrupting dimerization. By contrast, rather conservative substitutions at a valine on a different face of the alpha-helix disrupt dimerization, suggesting a high degree of specificity in the helix-helix interactions. This approach allows the interface between interacting helices to be defined.

A subdomain in the transmembrane domain is necessary for p185neu* activation.

The neu proto-oncogene encodes a protein highly homologous to the epidermal growth factor receptor. The neu protein (p185) has a molecular weight of 185,000 Daltons and, like the EGF receptor, possesses tyrosine kinase activity. neu is activated in chemically induced rat neuro/glioblastomas by substitution of valine 664 with glutamic acid within the transmembrane domain. The activated neu* protein (p185*) has an elevated tyrosine kinase activity and a higher propensity to dimerize, but the mechanism of this activation is still unknown. We have used site-directed mutagenesis to explore the role of specific amino acids within the transmembrane domain in this activation. We found that the lateral position and rotational orientation of the glutamic acid in the transmembrane domain does not correlate with transformation. However, the primary structure in the vicinity of Glu664 plays a significant role in this activation. Our results suggest that the Glu664 activation involves highly specific interactions in the transmembrane domain of p185.

Intramembrane helix-helix association in oligomerization and transmembrane signaling.

In spite of our greatly expanded knowledge of the primary structures of transbilayer receptor proteins, our knowledge of the tertiary and quaternary structures that define the biological activity of these receptors is scant. If we assume that the transmembrane regions of receptor proteins form stable alpha-helices regardless of the mechanism of insertion, a two-stage model of protein folding can be applied. For a multiple-helix protein, the two-stage model would predict that stable helical formation would be followed by an association of the helices to form the appropriate tertiary/quaternary structure. The two-stage model of protein folding is supported by various experiments with bacteriorhodopsin demonstrating that separate proteolytic fragments of bacteriorhodopsin can be refolded separately and can specifically recognize each other in order to associate and form a biologically active molecule. At the level of the bilayer, we propose that the energetics required for the association and packing of the helical transmembrane regions of a multiple-helix protein should not be significantly different from the association of separate single-helix proteins into an oligomer. Given the homogeneity in primary and secondary structure of the transmembrane regions of single-helix proteins, the association of multiple monomers may physically define high vs low affinity states and be a plausible mechanism of signal transduction. Increasing data suggest that oligomerization of receptor proteins may be involved in signal transduction. The transmembrane domains of receptor proteins appear to contain information critical to signaling and may be involved in a close contact site between receptors. This observation allows the two-stage model of protein folding for multiple-helix proteins to be directly applied to the oligomerization of single-helix receptor proteins. In addition, significant data suggest that the ectodomains and cytoplasmic domains are also involved in signaling and oligomerization of receptor molecules. In effect, the present data suggest that the most plausible model, both mechanistically and energetically, is one that includes both oligomerization and a global allosteric conformational change involving all of the defined domains of the receptor molecule. An oligomerization/conformational change model would predict that new sites of close contact would occur between the domains of the receptor molecule, some of which may be between the transmembrane helices. Therefore, experimenters should be able to generate peptides or small molecules that can specifically interfere with either the oligomerization or generation of new close-contact sites involved in the conformational change of the receptor that leads to signaling.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthetic peptides mimic the assembly of transmembrane glycoproteins.

The composition of the intramembranous domains of many receptors are remarkably uniform, yet there is evidence that many transmembrane proteins associate together to form specific noncovalent homo- or heterocomplexes within the membrane. We have synthesized peptides corresponding to transmembrane domains of glycophorin A, glycophorin C, and the interleukin 2-receptor Tac antigen to study the interactions between transmembrane domains in vitro. Synthetic transmembrane glycophorin A peptide formed a complex with native glycophorin and glycoproteins of erythrocyte and K562 cell membranes that was reversible, specific, and could be demonstrated in a natural bilayer system in the absence of detergents. Synthetic glycophorin C and interleukin 2-receptor Tac antigen transmembrane peptides, although similar in amino acid composition, did not interact with glycophorin and did not inhibit the binding of the synthetic glycophorin A transmembrane peptide to native glycophorin. It is proposed that the transmembrane segments of receptor proteins contain not only the structural information necessary for insertion and anchoring but specific binding sites that mediate interactions between transmembrane glycoproteins.

Thrombin-induced vimentin phosphorylation in cultured human umbilical vein endothelial cells.

Cultured human umbilical vein endothelial cells were stimulated with thrombin (1 unit/ml) for 15-30 s and then lysed with a solution of Triton X-100 containing [gamma-32P]adenosine triphosphate. Thrombin-stimulated human umbilical vein endothelial cells showed an enhanced incorporation of 32P into at least 12 different proteins as compared to control cells treated similarly. The observed enhanced phosphorylation required the active site of thrombin because diisopropylphosphoryl-thrombin had no effect on the level of phosphorylation. The molecular weight of one of the phosphoproteins was similar to that of the intermediate filament protein vimentin (55-60 kDa), a major protein in endothelial cells. This 59-kDa protein was Triton X-100-insoluble and reacted on a Western blot with antibody raised in guinea pig against Chinese hamster ovary cell vimentin. Addition of the anti-vimentin antibody to the thrombin-stimulated, phosphorylated lysate immuno-precipitated a single 32P-labeled protein (59 kDa). These results demonstrate that thrombin rapidly stimulates the phosphorylation of vimentin in cultured endothelial cells and links thrombin stimulation to the phosphorylation of a cytoskeletal protein.

Receptor-mediated activation of a phospholipase A2 in rabbit neutrophil plasma membrane.

Using the exogenous substrate [1-14C]oleate-labeled autoclaved Escherichia coli, we have demonstrated that the chemotactic factors fMet-Leu-Phe, complement component C5a, and leukotriene B4 [(5S,12R)-dihydroxy-6-cis,8-trans,11-trans,14-cis-icosatetraenoic acid] stimulate a phospholipase A2 of isolated plasma membranes of rabbit peritoneal neutrophils. Each of the chemotactic factors shows a biphasic concentration dependence with the optimal concentrations occurring at 1, 10, and 0.1 nM, respectively. The specific antagonists of fMet-Leu-Phe binding, carbobenzoxy-Phe-Met and t-butoxycarbonyl-Phe-Leu-Phe, effectively block the stimulation by fMet-Leu-Phe, indicating that the activation is receptor mediated. delta 6-trans-leukotriene [(5S-12R)-dihydroxy-all-trans-6,8,10,14-icosatetraenoic acid], a biologically inactive stereoisomer of leukotriene B4, does not stimulate phospholipase activity, suggesting that the enhancement by leukotriene B4 is also receptor mediated. The unstimulated and activated phospholipase exhibit a broad range of maximal activity between pH 7.0 and pH 8.5, both with an optimal pH of 8.5. The activation of the phospholipase by fMet-Leu-Phe is completely calcium dependent; no increase in activity is demonstrable if fMet-Leu-Phe is added in the absence of exogenous calcium or in the presence of EGTA. In contrast, the unstimulated plasma membrane activity of the phospholipase, as well as the activity arising after stimulation, is relatively insensitive to the concentration of calcium, being inhibited by less than 50% in the presence of 10 mM EGTA. The phospholipase hydrolyzes 1-[1-14C]palmitoyl-2-acyl-sn-glycerophosphoethanolamine to form only radioactive lysophosphatidylethanolamine as the product, indicating that the enzyme has an A2 specificity.

Chemotactic factors induced vimentin phosphorylation in rabbit peritoneal neutrophil.

Rabbit peritoneal neutrophils were reacted for 5-10 s with the chemotactic factors, fMet-Leu-Phe or (5S),(12R)-dihydroxy-6,8,11,14-(cis,trans,trans,cis)-eicosatetraenoic acid and then lysed with a solution of Triton X-100 and [gamma-32P]ATP. They showed an enhanced incorporation of 32P in Mr = 60,000- and 67,000-dalton polypeptides compared to control cells treated similarly. Another chemotactic factor, C5a, produced a similar but much lesser effect. The enhancement was not affected by the addition of the purified catalytic subunit of cAMP-dependent protein kinase, the inhibitor of cAMP-dependent protein kinase, or CaCl2, suggesting that the effect was not mediated by a cAMP-dependent or a Ca2+-dependent protein kinase. When analyzed by two-dimensional gel electrophoresis, the Mr = 60,000 phosphoprotein contained several phosphoproteins with different isoelectric points. The isoelectric point and molecular weight of one of them was similar to those of the intermediate filament protein, vimentin, purified from Chinese hamster ovary cells. Addition of the purified Chinese hamster ovary vimentin and [gamma-32P]ATP to the Triton X-100 lysate of fMet-Leu-Phe-treated neutrophils resulted as an enhanced incorporation of 32P into the vimentin. Addition of fMet-Leu-Phe to 32P-labeled intact neutrophils also enhanced incorporation of 32P into the vimentin of neutrophils. The results suggest that chemotactic factors stimulate vimentin phosphorylation in rabbit neutrophils.

Cyclic AMP receptor protein and cyclic AMP-dependent protein kinase activity in rabbit peritoneal neutrophils.

The cAMP receptor protein and cAMP-dependent protein kinase activity in rabbit peritoneal neutrophils have been identified. The cAMP receptor protein in either the plasma membrane or cytosol fractions, identified by photoaffinity labeling with 8-N3-[32P]cAMP, has an apparent molecular weight of 54,000. The cytosol and membrane receptor proteins have apparent dissociation constants for 8-N3-[32P]cAMP of 0.20 microM and 0.06 microM, respectively. The molecular weight and dissociation constant for 8-N3-[32P]cAMP of this cAMP receptor protein are similar to what has been known for RII, the regulatory subunit of the type II cAMP-dependent protein kinase. Unlike the human neutrophils, no evidence of RI activity was detected. cAMP-dependent protein kinase activity was identified by using histone as a substrate. Subcellular fractionation studies showed that the cAMP receptor protein and the cAMP-dependent protein kinase activity are most enriched in the cytosol fraction.