Chlorinated natural products include vancomycin and cryptophycin A. Their biosynthesis involves regioselective chlorination by flavin-dependent halogenases. We report the structural characterization of tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan. Tryptophan and flavin adenine dinucleotide (FAD) are separated by a 10 angstrom-long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. On the basis of biochemical studies, crystal structures, and by analogy with monooxygenases, we predict that FADH2 reacts with O2 to make peroxyflavin, which is decomposed by Cl-. The resulting HOCl is guided through the tunnel to tryptophan, where it is activated to participate in electrophilic aromatic substitution.

This study used functional magnetic resonance imaging to examine the effects of acute tryptophan (TRP) depletion (ATD), a well-recognized method for inducing transient cerebral serotonin depletion, on brain activity during probabilistic reversal learning. Twelve healthy male volunteers received a TRP-depleting drink or a balanced amino-acid drink (placebo) in a double-blind crossover design. At 5 h after drink ingestion, subjects were scanned while performing a probabilistic reversal learning task and while viewing a flashing checkerboard. The probabilistic reversal learning task enabled the separate examination of the effects of ATD on behavioral reversal following negative feedback and negative feedback per se that was not followed by behavioral adaptation. Consistent with previous findings, behavioral reversal was accompanied by significant signal change in the right ventrolateral prefrontal cortex (PFC) and the dorsomedial prefrontal cortex. ATD enhanced reversal-related signal change in the dorsomedial PFC, but did not modulate the ventrolateral PFC response. The ATD-induced signal change in the dorsomedial PFC during behavioral reversal learning extended to trials where subjects received negative feedback but did not change their behavior. These data suggest that ATD affects reversal learning and the processing of aversive signals by modulation of the dorsomedial PFC.

Cyanobacteriophage Syn9 is a large, contractile-tailed bacteriophage infecting the widespread, numerically dominant marine cyanobacteria of the genera Prochlorococcus and Synechococcus. Its 177,300 bp genome sequence encodes 226 putative proteins and six tRNAs. Experimental and computational analyses identified genes likely involved in virion formation, nucleotide synthesis, and DNA replication and repair. Syn9 shows significant mosaicism when compared with related cyanophages S-PM2, P-SSM2 and P-SSM4, although shared genes show strong purifying selection and evidence for large population sizes relative to other phages. Related to coliphage T4 - which shares 19% of Syn9's genes - Syn9 shows evidence for different patterns of DNA replication and uses homologous proteins to assemble capsids with a different overall structure that shares topology with phage SPO1 and herpes virus. Noteworthy bacteria-related sequences in the Syn9 genome potentially encode subunits of the photosynthetic reaction centre, electron transport proteins, three pentose pathway enzymes and two tryptophan halogenases. These genes suggest that Syn9 is well adapted to the physiology of its photosynthetic hosts and may affect the evolution of these sequences within marine cyanobacteria.

STATs (signal transducers and activators of transcription) are a family of transcription factors that are specifically activated to regulate gene transcription when cells encounter cytokines and growth factors. The crystal structure of an NH2-terminal conserved domain (N-domain) comprising the first 123 residues of STAT-4 was determined at 1.45 angstroms. The domain consists of eight helices that are assembled into a hook-like structure. The N-domain has been implicated in several protein-protein interactions affecting transcription, and it enables dimerized STAT molecules to polymerize and to bind DNA cooperatively. The structure shows that N-domains can interact through an extensive interface formed by polar interactions across one face of the hook. Mutagenesis of an invariant tryptophan residue at the heart of this interface abolished cooperative DNA binding by the full-length protein in vitro and reduced the transcriptional response after cytokine stimulation in vivo.

The PTPN22 locus is one of the strongest risk factors outside of the major histocompatability complex that associates with autoimmune diseases. PTPN22 encodes lymphoid protein tyrosine phosphatase (Lyp) which is expressed exclusively in immune cells. A single base change in the coding region of this gene resulting in an arginine to tryptophan amino acid substitution within a polyproline binding motif associates with type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosis, Hashimotos thyroiditis, Graves disease, Addison's disease, Myasthenia Gravis, vitiligo, systemic sclerosis juvenile idiopathic arthritis and psoriatic arthritis. Here, we review the current understanding of the PTPN22 locus from a genetic, geographical, biochemical and functional perspective.

The integral membrane protein diacylglycerol kinase (DGK) from Escherichia coli has been reversibly unfolded in a protein/detergent/mixed micelle system by varying the molar ratio of n-decyl beta-D-maltoside (DM) and sodium dodecyl sulfate (SDS). Unfolding was monitored by circular dichroism (CD) and ultraviolet (UV) absorbance spectroscopy. When unfolding is monitored by measuring changes in absorbance at 294 nm, two distinct denaturation phases are observed, indicative of a stable intermediate. When CD is used as a conformational probe, the resulting denaturation curve contains only one major transition, which corresponds to the first unfolding phase observed by absorbance changes. The unfolding behavior of several mutant proteins in which the tryptophan residues were selectively replaced made it possible to assign the first unfolding phase to a denaturation event in a cytoplasmic domain and the second phase to denaturation of the membrane-embedded portion of the protein. The denaturation curves fit well to a model which assumes two cooperative transitions and a linear relationship between unfolding free energy and SDS concentration. Extrapolation back to zero denaturant indicates an unfolding free energy of 6 kcal/mol for the cytoplasmic domain and 16 kcal/mol for the transmembrane domain. The high apparent stability of the transmembrane domain could explain the high degree of tolerance to amino acid substitutions seen for DGK and other membrane proteins. The approach described in this paper may be applicable to other membrane protein systems.

The tryptophanase structural gene, tnaA, of Escherichia coli K-12 was cloned and sequenced. The size, amino acid composition, and sequence of the protein predicted from the nucleotide sequence agree with protein structure data previously acquired by others for the tryptophanase of E. coli B. Physiological data indicated that the region controlling expression of tnaA was present in the cloned segment. Sequence data suggested that a second structural gene of unknown function was located distal to tnaA and may be in the same operon. The pattern of codon usage in tnaA was intermediate between codon usage in four of the ribosomal protein structural genes and the structural genes for three of the tryptophan biosynthetic proteins.

Benzoxazinoids are secondary metabolites that are effective in defence and allelopathy. They are synthesised in two subfamilies of the Poaceae and sporadically found in single species of the dicots. The biosynthesis is fully elucidated in maize; here the genes encoding the enzymes of the pathway are in physical proximity. This "biosynthetic cluster" might facilitate coordinated gene regulation. Data from Zea mays, Triticum aestivum and Hordeum lechleri suggest that the pathway is of monophyletic origin in the Poaceae. The branchpoint from the primary metabolism (Bx1 gene) can be traced back to duplication and functionalisation of the alpha-subunit of tryptophan synthase (TSA). Modification of the intermediates by consecutive hydroxylation is catalysed by members of a cytochrome P450 enzyme subfamily (Bx2-Bx5). Glucosylation by an UDP-glucosyltransferase (UGT, Bx8, Bx9) is essential for the reduction of autotoxicity of the benzoxazinoids. In some species 2,4-dihydroxy-1,4-benzoxazin-3-one-glucoside (DIBOA-glc) is further modified by the 2-oxoglutarate-dependent dioxygenase BX6 and the O-methyltransferase BX7. In the dicots Aphelandra squarrosa, Consolida orientalis, and Lamium galeobdolon, benzoxazinoid biosynthesis is analogously organised: The branchpoint is established by a homolog of TSA, P450 enzymes catalyse hydroxylations and at least the first hydroxylation reaction is identical in dicots and Poaceae, the toxic aglucon is glucosylated by an UGT. Functionally, TSA and BX1 are indole-glycerolphosphate lyases (IGLs). Igl genes seem to be generally duplicated in angiosperms. Modelling and biochemical characterisation of IGLs reveal that the catalytic properties of the enzyme can easily be modified by mutation. Independent evolution can be assumed for the BX1 function in dicots and Poaceae.

Human gammaD crystallin (HgammaD-Crys), a major protein of the human eye lens, is a primary component of cataracts. This 174-residue primarily beta-sheet protein is made up of four Greek keys separated into two domains. Mutations in the human gene sequence encoding HgammaD-Crys are implicated in early-onset cataracts in children, and the mutant protein expressed in Escherichia coli exhibits properties that reflect the in vivo pathology. We have characterized the unfolding, refolding, and competing aggregation of human wild-type HgammaD-Crys as a function of guanidinium hydrochloride (GuHCl) concentration at neutral pH and 37 degrees C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Wild-type HgammaD-Crys exhibited reversible refolding above 1.0 M GuHCl. The GuHCl unfolded protein was more fluorescent than its native counterpart despite the absence of metal or ion-tryptophan interactions. Aggregation of refolding intermediates of HgammaD-Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GuHCl, beyond the major conformational transition region. Atomic force microscopy of samples under aggregating conditions revealed the sequential appearance of small nuclei, thin protofibrils, and fiber bundles. The HgammaD-Crys fibrous aggregate species bound bisANS appreciably, indicating the presence of exposed hydrophobic pockets. The mechanism of HgammaD-Crys aggregation may provide clues to understanding age-onset cataract formation in vivo.

Most models of protein evolution are based upon proteins that form relatively rigid 3D structures. A significant fraction of proteins, the so-called disordered proteins, do not form rigid 3D structures and sample a broad conformational ensemble. Disordered proteins do not typically maintain long-range interactions, so the constraints on their evolution should be different than ordered proteins. To test this hypothesis, we developed and compared models of evolution for disordered and ordered proteins. Substitution matrices were constructed using the sequences of putative homologs for sets of experimentally characterized disordered and ordered proteins. Separate matrices, at three levels of sequence similarity (>85%, 85-60%, and 60-40%), were inferred for each type of protein structure. The substitution matrices for disordered and ordered proteins differed significantly at each level of sequence similarity. The disordered matrices reflected a greater likelihood of evolutionary changes, relative to the ordered matrices, and these changes involved nonconservative substitutions. Glutamic acid and asparagine were interesting exceptions to this result. Important differences between the substitutions that are accepted in disordered proteins relative to ordered proteins were also identified. In general, disordered proteins have fewer evolutionary constraints than ordered proteins. However, some residues like tryptophan and tyrosine are highly conserved in disordered proteins. This is due to their important role in forming protein-protein interfaces. Finally, the amino acid frequencies for disordered proteins, computed during the development of the matrices, were compared with amino acid frequencies for different categories of secondary structure in ordered proteins. The highest correlations were observed between the amino acid frequencies in disordered proteins and the solvent-exposed loops and turns of ordered proteins, supporting an emerging structural model for disordered proteins.

Tyrosine hydroxylase (TyrOH) catalyzes the conversion of tyrosine to L-DOPA, the rate-limiting step in the biosynthesis of the catecholamines dopamine, adrenaline, and noradrenaline. TyrOH is highly homologous in terms of both protein sequence and catalytic mechanism to phenylalanine hydroxylase (PheOH) and tryptophan hydroxylase (TrpOH). The crystal structure of the catalytic and tetramerization domains of TyrOH reveals a novel alpha-helical basket holding the catalytic iron and a 40 A long anti-parallel coiled coil which forms the core of the tetramer. The catalytic iron is located 10 A below the enzyme surface in a 17 A deep active site pocket and is coordinated by the conserved residues His 331, His 336 and Glu 376. The structure provides a rationale for the effect of point mutations in TyrOH that cause L-DOPA responsive parkinsonism and Segawa's syndrome. The location of 112 different point mutations in PheOH that lead to phenylketonuria (PKU) are predicted based on the TyrOH structure.

Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5' of uridylates of RNAs. Blocking either the 5' or 3' terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn(2+) at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg(2+) and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn(2+) needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.

Amino acids (AA) were traditionally classified as nutritionally essential or nonessential for animals and humans based on nitrogen balance or growth. A key element of this classification is that all nonessential AA (NEAA) were assumed to be synthesized adequately in the body as substrates to meet the needs for protein synthesis. Unfortunately, regulatory roles for AA in nutrition and metabolism have long been ignored. Such conceptual limitations were not recognized until recent seminal findings that dietary glutamine is necessary for intestinal mucosal integrity and dietary arginine is required for maximum neonatal growth and embryonic survival. Some of the traditionally classified NEAA (e.g. glutamine, glutamate, and arginine) play important roles in regulating gene expression, cell signaling, antioxidative responses, and immunity. Additionally, glutamate, glutamine, and aspartate are major metabolic fuels for the small intestine and they, along with glycine, regulate neurological function. Among essential AA (EAA), much emphasis has been placed on leucine (which activates mammalian target of rapamycin to stimulate protein synthesis and inhibit proteolysis) and tryptophan (which modulates neurological and immunological functions through multiple metabolites, including serotonin and melatonin). A growing body of literature leads to a new concept of functional AA, which are defined as those AA that regulate key metabolic pathways to improve health, survival, growth, development, lactation, and reproduction of organisms. Both NEAA and EAA should be considered in the classic "ideal protein" concept or formulation of balanced diets to maximize protein accretion and optimize health in animals and humans.

BACKGROUND

Major depression is a common disorder but the pathophysiology is poorly understood. Current hypotheses implicate deficient function of brain serotonin pathways because drugs that selectively increase brain serotonin activity are effective antidepressants. However, there is no direct evidence that lowered serotonin function causes major depression. We aimed to assess whether lowering of brain serotonin activity by depletion of its amino acid precursor, tryptophan, could provoke a short-term relapse of clinically significant symptoms in women vulnerable to major depressive disorder.

METHODS

We studied 15 women who had suffered recurrent episodes of major depression but had recovered and were no longer on drug treatment. Patients received two amino acid mixtures in a double-blind crossover design. One of the mixtures was nutritionally balanced and contained tryptophan and the other was identical except it contained no tryptophan. Participants were scored on the Hamilton rating scale for depression (HAMD) before and 7 h after drinking each mixture. They also completed hourly self-rated measures of mood during this period. Blood samples were also taken at baseline and 7 h for measurement of plasma tryptophan.

RESULTS

The tryptophan-free mixture produced a 75% reduction in plasma tryptophan concentration. After drinking the tryptophan-free mixture, ten of the 15 women experienced temporary but clinically significant depressive symptoms. The mean difference in total HAMD scores (7 h minus baseline) were significantly higher after the tryptophan-free mixture than after the nutritionally balanced mixture (7.3 vs 0.15 [95% CI 4.5-9.9]; p < 0.001). No changes in mood were seen after taking the nutritionally balanced mixture.

CONCLUSIONS

We conclude that rapid lowering of brain serotonin function can precipitate clinical depressive symptoms in well, untreated individuals who are vulnerable to major depressive disorder. The findings support a key role for deficient serotonin function in the aetiology of depression.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor recognized for its role in xenobiotic metabolism. The physiologic function of AHR has expanded to include roles in immune regulation, organogenesis, mucosal barrier function, and the cell cycle. These functions are likely dependent upon ligand-mediated activation of the receptor. High-affinity ligands of AHR have been classically defined as xenobiotics, such as polychlorinated biphenyls and dioxins. Identification of endogenous AHR ligands is key to understanding the physiologic functions of this enigmatic receptor. Metabolic pathways targeting the amino acid tryptophan and indole can lead to a myriad of metabolites, some of which are AHR ligands. Many of these ligands exhibit species selective preferential binding to AHR. The discovery of specific tryptophan metabolites as AHR ligands may provide insight concerning where AHR is activated in an organism, such as at the site of inflammation and within the intestinal tract.

Although amino acid auxotrophs are among the most frequently isolated mutations in microorganisms, no mutants that require amino acids have been isolated at the whole plant level. Tryptophan-requiring mutants of the cruciferous plant Arabidopsis thaliana have now been isolated by selecting for resistance to 5-methylanthranilic acid. The tryptophan requirement of one mutant, trpl-1, results from a defect in the second step of the tryptophan pathway catalyzed by anthranilate phosphoribosyl transferase. Mutant trpl-1 plants are highly fluorescent and aromatic because they accumulate anthranilic acid and anthranilate beta-glucoside. Plants homozygous for the trpl-1 mutation exhibit a syndrome of morphological defects suggestive of a defect in the biosynthesis, metabolism, or localization of a tryptophan derivative such as auxin. All of these morphological phenotypes cosegregate with the tryptophan requirement as a simple Mendelian recessive trait.

Nicotinic acetylcholine receptors contain a readily reducible disulfide bond at the periphery of the acetylcholine binding site. Following reduction of this disulfide, the binding site is susceptible to affinity labeling by electrophilic reagents with quaternary ammonium moieties. We reduced purified receptor from Torpedo californica electric tissue and affinity alkylated it with 4-(N-maleimido)benzyltri[3H]methylammonium iodide. The label was incorporated solely into the alpha subunit of the receptor. Isolated, labeled alpha subunit was cleaved with CNBr, and the fragments were separated by reverse-phase high-performance liquid chromatography. A uniquely labeled CNBr fragment was isolated, and its partial sequence was determined by automated Edman degradation. This CNBr fragment was cleaved at tryptophan residues, the subfragments were separated, and the labeled subfragments were partially sequenced. From our protein sequence information, we identify the labeled CNBr fragment as residues 179 to 207 of the sequence of alpha predicted from the cDNA sequence (Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., Asai, M., Inayama, S., Miyata, T., and Numa, S. (1982) Nature (Lond.) 299, 793-797). From the cycle of the Edman degradation in which radioactive residues are released, we conclude that Cys 192 and, possibly in addition, Cys 193 are the residues specifically labeled by 4-(N-maleimido)benzyltri[3H]methylammonium iodide. They are, therefore, close to the acetylcholine binding site.

Recent studies have indicated that a newly identified second isoform of the tryptophan hydroxylase gene (TPH2) is preferentially involved in the rate-limiting synthesis of neuronal serotonin. Genetic variation in the human TPH2 gene (hTPH2) has been associated with altered in vitro enzyme activity as well as increased risk for mood disorders. Here, we provide the first in vivo evidence that a relatively frequent regulatory variant (G(-844)T) of hTPH2 biases the reactivity of the amygdala, a neural structure critical in the generation and regulation of emotional behaviors.

We have determined the complete amino acid sequence of beta 2-glycoprotein I (Mr, congruent to 50,000), a human plasma protein that is associated with lipids and binds to platelets but whose function is not yet known. The protein consists of 326 amino acids and has five attached glucosamine-containing oligosaccharides. The protein is rich in cysteine and proline, and the sequence is notable for the frequent occurrence of Cys-Pro linkages at regular intervals. Computerized analysis of the sequence reveals five consecutive homologous segments in which cysteine, proline, and tryptophan appear to be highly conserved. This suggests that beta 2-glycoprotein I may have evolved by repeated duplications of a gene coding for a 60-amino acid segment of protein.

To better understand ligand-induced structural transitions in cytochrome P450 2B4, protein-ligand interactions were investigated using a bulky inhibitor. Bifonazole, a broad spectrum antifungal agent, inhibits monooxygenase activity and induces a type II binding spectrum in 2B4dH(H226Y), a modified enzyme previously crystallized in the presence of 4-(4-chlorophenyl)imidazole (CPI). Isothermal titration calorimetry and tryptophan fluorescence quenching indicate no significant burial of protein apolar surface nor altered accessibility of Trp-121 upon bifonazole binding, in contrast to recent results with CPI. A 2.3 A crystal structure of 2B4-bifonazole reveals a novel open conformation with ligand bound in the active site, which is significantly different from either the U-shaped cleft of ligand-free 2B4 or the small active site pocket of 2B4-CPI. The O-shaped active site cleft of 2B4-bifonazole is widely open in the middle but narrow at the top. A bifonazole molecule occupies the bottom of the active site cleft, where helix I is bent approximately 15 degrees to accommodate the bulky ligand. The structure also defines unanticipated interactions between helix C residues and bifonazole, suggesting an important role of helix C in azole recognition by mammalian P450s. Comparison of the ligand-free 2B4 structure, the 2B4-CPI structure, and the 2B4-bifonazole structure identifies structurally plastic regions that undergo correlated conformational changes in response to ligand binding. The most plastic regions are putative membrane-binding motifs involved in substrate access or substrate binding. The results allow us to model the membrane-associated state of P450 and provide insight into how lipophilic substrates access the buried active site.

BACKGROUND

The late embryogenesis abundant (LEA) proteins cover a number of loosely related groups of proteins, originally found in plants but now being found in non-plant species. Their precise function is unknown, though considerable evidence suggests that LEA proteins are involved in desiccation resistance. Using a number of statistically-based bioinformatics tools the classification of a large set of LEA proteins, covering all Groups, is reexamined together with some previous findings. Searches based on peptide composition return proteins with similar composition to different LEA Groups; keyword clustering is then applied to reveal keywords and phrases suggestive of the Groups' properties.

RESULTS

Previous research has suggested that glycine is characteristic of LEA proteins, but it is only highly over-represented in Groups 1 and 2, while alanine, thought characteristic of Group 2, is over-represented in Group 3, 4 and 6 but under-represented in Groups 1 and 2. However, for LEA Groups 1 2 and 3 it is shown that glutamine is very significantly over-represented, while cysteine, phenylalanine, isoleucine, leucine and tryptophan are significantly under-represented. There is also evidence that the Group 4 LEA proteins are more appropriately redistributed to Group 2 and Group 3. Similarly, Group 5 is better found among the Group 3 LEA proteins.

CONCLUSIONS

There is evidence that Group 2 and Group 3 LEA proteins, though distinct, might be related. This relationship is also evident in the overlapping sets of keywords for the two Groups, emphasising alpha-helical structure and, at a larger scale, filaments, all of which fits well with experimental evidence that proteins from both Groups are natively unstructured, but become structured under stress conditions. The keywords support localisation of LEA proteins both in the nucleus and associated with the cytoskeleton, and a mode of action similar to chaperones, perhaps the cold shock chaperones, via a role in DNA-binding. In general, non-globular and low-complexity proteins, such as the LEA proteins, pose particular challenges in determining their functions and modes of action. Rather than masking off and ignoring low-complexity domains, novel tools and tool combinations are needed which are capable of analysing such proteins in their entirety.

BACKGROUND

Mdm-2, a zinc finger protein, negatively regulates the p53 tumor suppressor gene product by binding to it and preventing transcriptional activation (16).

METHODS

Assays for p53 mediated transcription, repression and activation by mutant and wild-type p53 proteins were used to measure the ability of mdm-2 to block each activity.

RESULTS

Mdm-2 was able to inhibit all three functions of the wild-type and mutant p53 activities; transcriptional activation by the wild-type protein, transcriptional activation by the mutant p53 protein, and repression by the wild-type protein.

CONCLUSIONS

The mdm protein binds to the amino terminal portion of the p53 protein and, in so doing, blocks the ability of p53 to interact with the transcriptional machinery of the cell (23). The mdm-2 protein binds to both leucine-tryptophan residues at amino acids 22 and 23, from the amino terminal end of the protein, and in so doing, prevents all p53 functions. The ability of a mutant p53 protein to transactivate a multidrug resistance-1 gene promoter is blocked by mdm-2 and the ability of the wild-type p53 protein to repress transcription of some genes is also blocked by the mdm-2 protein. Thus, all three functions of the p53 protein-transcriptional activation, repression and mutant protein activation-require the p53 amino terminal domain functions and are regulated by the mdm-2 protein in a cell. When mdm-2 is overproduced, resulting in a tumor or transformation of a cell, all of the p53 activities are inactivated.

The pleckstrin homology (PH) domain is an approximately 100-amino-acid region of sequence homology present in numerous proteins of diverse functions, which forms a discrete structural module. Several ligands capable of binding to PH domain-containing proteins have been identified including phosphatidylinositol 4,5-bisphosphate (PIP2) and the G beta gamma subunits of heterotrimeric G proteins (G beta gamma), which bind to the amino and carboxyl termini of the PH domain, respectively. Here we report that the binding of G beta gamma and lipid to the PH domain of the beta-adrenergic receptor kinase (beta ARK) synergistically enhances agonist-dependent receptor phosphorylation and that both PH domain-binding ligands are required for membrane association of the kinase. PIP2 and to a lesser extent phosphatidylinositol 4-phosphate, phosphatidylinositol, and phosphatidic acid were the only lipids tested capable, in the presence of G beta gamma, of enhancing beta ARK activity. In contrast, the Km and Vmax for phosphorylation of a soluble beta ARK substrate (casein) was not altered in either the presence or absence of G beta gamma and/or PIP2. A fusion protein of the beta ARK containing an intact PH domain inhibits G beta gamma/PIP2-dependent beta ARK activity. In contrast, a mutant fusion protein in which a tryptophan residue, invariant in all PH domain sequences, is mutated to alanine shows no inhibitory activity. The requirement for the simultaneous presence of two PH domain binding ligands represents a previously unappreciated mechanism for effecting membrane localization of a protein and may have relevance to other PH domain-containing proteins.