Protein tyrosine phosphatase inhibitors in Fc gamma RI-induced myeloid oxidant signaling

Fc-receptor stimulation in myeloid cells results in increased oxygen consumption, termed the respiratory burst, which is coupled to a rapid and transient increase in tyrosine phosphorylation of cellular proteins. In a previous paper in this journal we showed that the protein tyrosine phosphatase (PTPase) inhibitors sodium orthovanadate and phenylarsine oxide (PAO) block the Fc gamma RI-induced respiratory burst in interferon-gamma-differentiated U937 cells (U937IF) while augmenting the Fc gamma RI-induced tyrosine phosphorylation of cellular proteins. Herein we examine the effects of PTPase inhibitors on specific molecules involved in Fc gamma RI signaling. We show that orthovanadate and PAO augmented the Fc gamma RI-induced tyrosine phosphorylation of the adaptor protein CBL. CBL interactions with other phosphoproteins, among them SHC and CRKL, were also augmented in response to pretreatment with the PTPase inhibitors. SHC was tyrosine phosphorylated in response to Fc gamma RI stimulation of U937IF cells and bound to the SH2 domain of GRB2 in a stimulation-dependent manner. In fusion protein pull-down experiments the interaction of SHC with the SH2 domain of GRB2 was increased in PTPase inhibitor pretreated U937IF cells in response to Fc gamma RI stimulation. Our data support the hypothesis that a tyrosine dephosphorylation event is required for effective transmission of the Fc gamma RI signal to result in activation of the myeloid respiratory burst response.     

Thyroid metastasis from rectal carcinoma coexisting with Hashimoto''s thyroiditis: gray-scale and power Doppler sonographic findings

BACKGROUND: Interferon (IFN) alpha and IFN gamma have synergistic in vitro and in vivo effects and have each been used in the treatment of anogenital condylomata acuminata. STUDY GOALS: To compare the efficacy and safety of regional 3 x 10(6) U IFN alpha (Group A), 3 x 10(6) U IFN gamma (Group B), 1.5 x 10(6) U IFN alpha plus 1.5 x 10(6) U IFN gamma (Group C), and 3 x 10(6) U IFN alpha plus 1.5 x 10(6) U IFN gamma (Group D) in the treatment of recalcitrant anogenital condylomata acuminata. STUDY DESIGN: Six-week courses of regional IFNs were administered in a randomized, double-blind, multicenter study. Response was assessed as change in the total area affected by condylomata. RESULTS: The treatments of Groups A, B, and C were similarly effective (complete response in 13.6%, 18.5%, and 16.0%, respectively). Group D had the lowest rate of complete response (3.8%), but this combination was the most effective when partial and complete responses were combined (73.0%). CONCLUSION: Regional IFN is a moderately effective and safe treatment in patients with recalcitrant anogenital condylomata acuminata. Combinations of IFNs alpha and gamma were not superior to IFN monotherapy.     

Sites other than nucleotide 234 determine cardiovirulence in natural isolates of coxsackievirus B3

The genetic site(s) that naturally determine the cardiovirulence phenotype of coxsackievirus B3 (CVB3) have yet to be mapped. Using two closely related CVB3 strains that differed in terms of cardiovirulence phenotype in mice, we previously reported the difference in phenotype mapped to a single site, nucleotide 234 (nt234) in the 5' non-translated region (NTR) of the CVB3 genome. When nt234 was C, the virus was attenuated and when U, the virus was cardiovirulent. To determine whether this finding was applicable to other strains of CVB3, we examined 13 different naturally occurring CVB3 strains isolated in different years in the United States. We determined that only two isolates induced severe inflammatory heart muscle disease in C3H/HeJ male mice. Using PCR products as sequencing templates, we determined the 5' NTR sequence from each viral genome. Alignment of these sequences and other published CVB3 5' NTR sequences suggests as many as four separate lineages, with commonly used laboratory strains clustering closely in one branch. An examination of the sequences showed that regardless of cardiovirulence phenotype, nt234 was invariably uridine. Thus, the previously reported cytidine at nt234 is most likely the result of a rare mutation and is not a naturally occurring variation and other sites must account for the variance in virulence seen in natural isolates of CVB3.     

A molecular switch changes the signalling pathway used by the Fc gamma RI antibody receptor to mobilise calcium

BACKGROUND: Leukocytes express Fc gamma receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody-directed cellular cytotoxicity. The nature of the myeloid response to Fc gamma receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability. RESULTS: We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fc gamma receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor Fc gamma RI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the gamma chain, an Fc gamma RI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of Fc gamma RI resulted in the Fc gamma RIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated. CONCLUSIONS: The switch in Fc gamma RI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by Fc gamma RI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.     

Differential interaction of Crkl with Cbl or C3G, Hef-1, and gamma subunit immunoreceptor tyrosine-based activation motif in signaling of myeloid high affinity Fc receptor for IgG (Fc gamma RI)

Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following Fc gamma RI aggregation in U937IF cells. Fc gamma RI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing gamma subunit of Fc gamma RI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and Fc gamma RI gamma after Fc gamma RI activation and the constitutive association between C3G and Crkl provide the first evidence that a Fc gamma RI gamma-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.     

U1 snRNA is cleaved by RNase III and processed through an Sm site-dependent pathway

Core snRNP proteins bind snRNA through the conserved Sm site, PuA(U)n>/=3GPu. While yeast U1 snRNA has three matches to the Sm consensus, the U1 3'-terminal Sm site was found to be both necessary and sufficient for U1 function. Mutation of this site inhibited pre-mRNA splicing, blocked cell division and resulted in the accumulation of two 3'-extended forms of the U1 snRNA. Cells which harbor the Sm site mutation lack mature U1 RNA (U1alpha) but have a minor polyadenylated species, U1gamma, and a prominent, non-polyadenylated species, U1beta. Metabolic depletion of the essential Sm core protein, Smd1p, also resulted in the increased accumulation of U1beta and U1gamma. In vitro, synthetic U1 precursors were cleaved by Rnt1p (RNase III) very near the U1beta 3'-end observed in vivo. We propose that U1beta is an Rnt1p-cleaved intermediate and that U1 maturation to the U1alpha form occurs through an Sm-sensitive step. Interestingly, both U1alpha and a second, much longer RNA, U1straightepsilon, were produced in an rnt1 mutant strain. These results suggest that yeast U1 snRNA processing may progress through Rnt1p-dependent and Rnt1p-independent pathways, both of which require a fun-ctional Sm site for final snRNA maturation.     

Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains

The lactose permease of Escherichia coli is a membrane transport protein containing 12 transmembrane hydrophobic domains connected by hydrophilic loops. Coexpression of lacY gene fragments encoding contiguous polypeptides corresponding to the first and second halves of the permease [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325-4329] or the first two transmembrane domains and the remainder of the molecule [Wrubel, W., Stochaj, U., Sonnewald, U., Theres, C., & Ehring, R. (1990) J. Bacteriol. 172, 5374-5381] leads to active lactose transport. It is shown here that contiguous permease fragments with discontinuities in loop 1 (periplasmic), loop 6 (cytoplasmic), or loop 7 (periplasmic) exhibit transport activity; however, fragments with discontinuities in transmembrane domains III or VII fail to do so. The results are consistent with the interpretation that contiguous permease fragments with discontinuities in hydrophilic loops form functional duplexes, while fragments with discontinuities in transmembrane alpha-helical domains do not. On the basis of this notion, a series of contiguous, nonoverlapping permease fragments with discontinuities at various positions in loop 6, putative helix VII, and loop 7 were coexpressed to approximate the boundaries of putative transmembrane domain VII. Contiguous fragments with a discontinuity between Leu222 and Trp223 or between Gly254 and Glu255 are functional, but fragments with a discontinuity between Cys234 and Thr235, between Gln241 and Gln242, or between Phe247 and Thr248 are inactive. Therefore, it is likely that Leu222 and Gly254 are located in hydrophilic loops 6 and 7, respectively, while Cys234, Gln241, and Phe247 are probably located within transmembrane domain VII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracluster restriction of Fc receptor gamma-chain tyrosine phosphorylation subverted by a protein-tyrosine phosphatase inhibitor

This study shows that aggregation of U937 cell high affinity IgG Fc receptor (Fc gamma RI) results in the transient tyrosine phosphorylation of Fc gamma RI gamma-chain but not the phosphorylation of gamma-chains associated with nonaggregated IgA Fc receptors (Fc alpha R) on the same cells. Thus, normally, tyrosine phosphorylation of gamma-chains is limited to FcR in aggregates. In contrast, aggregation of Fc gamma RI in the presence of vanadate induced the sustained tyrosine phosphorylation of Fc gamma RI gamma-chains and the rapid and extensive phosphorylation of nonaggregated Fc alpha R gamma-chains and low affinity IgG Fc receptors (Fc gamma RII). This global phosphorylation of motifs on nonaggregated FcR was also detected upon aggregation of Fc alpha R or Fc gamma RII, which induced the phosphorylation of nonaggregated Fc gamma RI gamma-chains. Vanadate prevented dephosphorylation of proteins and increased kinase activity in stimulated cells. Evidence failed to support alternative explanations such as acquisition of phospho-gamma through subunit exchange or a coalescence of nonaggregated with aggregated FcR. It is likely, therefore, that activated kinases interacted with nonaggregated FcR in stimulated cells. Pervanadate induced the tyrosine phosphorylation of gamma-chains in the absence of FcR cross-linking, indicating that the kinases could be activated by phosphatase inhibition and could react with nonaggregated substrates. We conclude that under normal conditions there is a vanadate-sensitive mechanism that prevents tyrosine phosphorylation of nonaggregated FcR gamma-chain motifs in activated cells, restricting their phosphorylation to aggregates.     

Three-dimensional structure of the ion-channel forming peptide trichorzianin TA VII bound to sodium dodecyl sulfate micelles

Trichorzianin TA VII, Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the alpha, alpha-dialkylated amino acids, alpha-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. 1H sequential assignment have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, 1H-13C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as 3JNHC alpha H coupling constants, temperature coefficients of amide protons (delta gamma/delta TNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances six phi dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 A for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1-8 and 11-19, linked by a beta-turn that leads to an angle about 90 degrees-100 degrees between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations.     

Metal-catalyzed oxidation and mutagenesis studies on the iron(II) binding site of 1-aminocyclopropane-1-carboxylate oxidase

The final step in the biosynthesis of the plant signaling molecule ethylene is catalyzed by 1-aminocyclopropane-1-carboxylate (ACC) oxidase, a member of the non-heme iron(II) dependent family of oxygenases and oxidases, which has a requirement for ascorbate as a co-substrate and carbon dioxide as an activator. ACC oxidase (tomato) has a particularly short half-life under catalytic conditions undergoing metal-catalyzed oxidative (MCO) fragmentation. Sequence comparisons of ACC oxidases with isopenicillin N synthase (IPNS) and members of the 2-oxoglutarate Fe(II) dependent dioxygenases show an aspartate and two of six ACC oxidase conserved histidine residues are completely conserved throughout this subfamily of Fe(II) dependent oxygenases/oxidases. Previous mutagenesis, spectroscopic, and crystallographic studies on IPNS indicate that the two completely conserved histidine and aspartate residues act as Fe(II) ligands. To investigate the role of the conserved aspartate and histidine residues in ACC oxidase (tomato fruit), they were substituted via site-directed mutagenesis. Modified ACC oxidases produced were H39Q, H56Q, H94Q, H177Q, H177D, H177E, D179E, D179N, H177D&D179E, H211Q, H234Q, H234D, and H234E. Among those histidine mutants replaced by glutamine, H39Q, H56Q, H94Q, and H211Q were catalytically active, indicating these histidines are not essential for catalysis. Mutant enzymes H177D, H177Q, D179N, H177D&D179E, H234Q, H234D, and H234E were catalytically inactive consistent with the assignment of H177, D179, and H234 as iron ligands. Replacement of H177 with glutamate or D179 with glutamate resulted in modified ACC oxidases which still effected the conversion of ACC to ethylene, albeit at a very low level of activity, which was stimulated by bicarbonate. The H177D (inactive), H177E (low activity), D179E (low activity), and H234Q (inactive) modified ACC oxidases all underwent MCO fragmentation, indicating that they can bind iron, dioxygen, ACC, and ascorbate. The results suggest that MCO cleavage results from active site-mediated reactions and imply that, while H177, D179, and H234 are all involved in metal ligation during catalysis, ligation to H234 is not required for fragmentation. It is possible that MCO fragmentation results from reaction of incorrectly folded or "primed" ACC oxidase.     

The null mutant of the U(L)31 gene of herpes simplex virus 1: construction and phenotype in infected cells

Earlier studies have shown that the U(L)31 protein is homogeneously distributed throughout the nucleus and cofractionates with nuclear matrix. We report the construction from an appropriate cosmid library a deletion mutant which replicates in rabbit skin cells carrying the U(L)31 gene under a late (gamma1) viral promoter. The mutant virus exhibits cytopathic effects and yields 0.01 to 0.1% of the yield of wild-type parent virus in noncomplementing cells but amounts of virus 10- to 1,000-fold higher than those recovered from the same cells 3 h after infection. Electron microscopic studies indicate the presence of small numbers of full capsids but a lack of enveloped virions. Viral DNA extracted from the cytoplasm of infected cells exhibits free termini indicating cleavage/packaging of viral DNA from concatemers for packaging into virions, but analyses of viral DNAs by pulsed-field electrophoresis indicate that at 16 h after infection, both the yields of viral DNA and cleavage of viral DNA for packaging are decreased. The repaired virus cannot be differentiated from the wild-type parent. These results suggest the possibility that U(L)31 protein forms a network to enable the anchorage of viral products for the synthesis and/or packaging of viral DNA into virions.     

Interferon-gamma inhibition of human thyrotropin receptor gene expression

To evaluate the role of interferon-gamma (IFN gamma) on human thyroid-specific gene expression, the effect of IFN gamma on TSH- and cAMP-induced TSH receptor gene expression was studied using cultured thyroid cells obtained from normal thyroid glands and those from patients with Graves' disease. Incubation of Graves' thyroid cells with 1.0 U/L bovine TSH or 1.0 mM 8-bromo-cAMP resulted in a 2-fold increase in TSH receptor mRNA expression, which was markedly inhibited in the presence of IFN gamma in a dose- and time-dependent manner. This inhibitory effect was completely neutralized by monoclonal antibody against IFN gamma. IFN alpha and -beta had no influence on TSH- and cAMP-stimulated TSH receptor mRNA expression. Paranodular normal thyroid cells showed the same results as those obtained using Graves' thyroid cells. Scatchard analysis of the [125I]TSH binding study showed that IFN gamma inhibited the number of TSH receptors up-regulated by TSH on the cell surface at the low affinity binding site (4.1 vs. 8.2 x 10(5)/cell). These results indicate that IFN gamma suppresses TSH- and cAMP stimulated human TSH receptor gene expression, resulting in a decrease in the number of TSH receptors. In conclusion, IFN gamma interacts via an intermediate pathway of TSH signal transduction and attenuates TSH receptor synthesis in normal and Graves' thyroid cells.     

Bolus dispersal through the lungs in surfactant replacement therapy

A model is presented of surfactant replacement therapy. An instilled bolus is pushed into the lungs on the first inspiration, coating the airways with a layer of surfactant and depositing some in the alveoli. Layer thickness depends on the capillary number (muU/gamma, where mu, U, and gamma are bolus viscosity, advancing meniscus velocity, and surface tension, respectively). Larger capillary number leads to thicker layers, reducing alveolar delivery. Subsequently, surface tension gradients sweep surfactant into alveoli not receiving surfactant during the first inspiration. The effects on spreading of sorption kinetics, bolus viscosity, initial layer thickness, initial penetration of surfactant, gravity, and shear stress are examined. Sorption nearly eliminates surface tension gradients in central airways but produces a sharp transition at the leading edge of the exogenous layer. Local thinning of the liquid layer results, trapping 95% of the surfactant in the airways. Gravity and ventilation augment transport somewhat. Transport to the periphery takes 4-170 s for the leading edge but considerably longer for the bulk of the surfactant. The model demonstrates how the various physical parameters governing surfactant distribution might alter the response to surfactant replacement therapy.     

Site-directed mutagenesis of beta-lactamase TEM-1. Investigating the potential role of specific residues on the activity of Pseudomonas-specific enzymes

From sequence alignments, two groups can be defined for the carbenicillin-hydrolysing beta-lactamases (CARB enzymes). One group includes the Pseudomonas-specific enzymes PSE-1, PSE-4, CARB-3, CARB-4 and also the Proteus mirabilis GN79, for which the well-conserved residue Lys 234 in all class-A beta-lactamases is changed to an arginine residue. The second group includes the enzymes PSE-3 and AER-1 which have an arginine or a lysine residue at position 165. All these enzymes also have leucine at position 68, threonine at position 104 and glycine at position 240. We engineered these mutations into the TEM-1 beta-lactamase to study their potential role in defining the substrate profile of the CARB enzymes. The mutations K234R and E240G in TEM-1 noticeably increased the hydrolysis of carboxypenicillins relative to other penicillins by approximately sixfold and twofold, respectively. The variant E240G also demonstrated an improved rate of second-generation cephalosporin and cefotaxime hydrolysis. In contrast, the substitution of Trp165 by arginine does not extend the substrate profile to alpha-carboxypenicillins nor does it noticeably modify the kinetic behavior of the enzyme. The mutations M68L and E104T do not have a large effect on the hydrolysis rate but the mutation E104T enhances the affinity of the enzyme for third-generation cephalosporins. As the mutation K234R resulted in a severe decrease in the affinity for carboxypenicillins, the double mutant E240G/K234R was constructed in an attempt to enhance the CARB character of the enzyme. Contrary to what could be expected, the additional mutation E240G for the TEM-1 K234R enzyme increases neither the catalytic constant for the carboxypenicillins nor the affinity towards these substrates. Consequently, this study strongly suggests that the three-dimensional structures of the active site of the TEM-1 enzyme and PSE-3, PSE-4 or other related enzymes are significantly different. This probably explains the discrepancy of the substrate profile between the CARB enzymes and the TEM-1 protein variants.     

The gamma subunit of the Na,K-ATPase induces cation channel activity

The gamma subunit of the Na,K-ATPase is a hydrophobic protein of approximately 10 kDa. The gamma subunit was expressed in Sf-9 insect cells and Xenopus oocytes to ascertain its role in Na,K-ATPase function. Immunoblotting has shown that the gamma subunit is expressed in Sf-9 cells infected with recombinant baculovirus containing the cDNA for the human gamma subunit. Confocal microscopy demonstrates that the gamma subunit can be delivered to the plasma membrane of Sf-9 cells independently of the other Na,K-ATPase subunits and that gamma colocalizes with alpha1 when these proteins are coexpressed. When Sf-9 cells were coinfected with alpha1 and gamma, antibodies to the gamma subunit were able to coimmunoprecipitate the alpha1 subunit, suggesting that gamma is able to associate with alpha1. The gamma subunit is a member of a family of single-pass transmembrane proteins that induces ion fluxes in Xenopus oocytes. Evidence that the gamma subunit is a functional component was supported by experiments showing gamma-induced cation channel activity when expressed in oocytes and increases in Na+ and K+ uptake when expressed in Sf-9 cells.     

Phosphorylation of the gamma subunit of the retinal photoreceptor cGMP phosphodiesterase by the cAMP-dependent protein kinase and its effect on the gamma subunit interaction with other proteins

Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.