Vascularity and tendon pathology in the rotator cuff: a review of literature and implications for rehabilitation and surgery.

OBJECTIVE: To compile histological and imaging research detailing the microvascularity of the rotator cuff and determine the clinical application of these findings for clinicians. METHODS: A computer-assisted literature search of MEDLINE (1966 to September 2008) using keywords related to blood flow to the shoulder and limited to humans and English language. A hand search was also performed by three of the authors. RESULTS: Nineteen studies met inclusion and exclusion criteria. CONCLUSIONS: The relationship between the variables of vascularity, age and degeneration remains unclear. Recent studies with stronger design and better technology support the fact that increased vascularity is a normal response to smaller tears, but that as tear size increases the healing response fails and decreased vascularity is observed. Also, impingement may cause hypovascularity. These studies support the possibility that people without symptoms may have normal blood flow even with ageing. Finally, exercise may increase blood flow to the rotator cuff. These findings have both surgical and rehabilitation implications.

Receptor-G protein gamma specificity: gamma11 shows unique potency for A(1) adenosine and 5-HT(1A) receptors.

G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G proteins), which consist of an alpha subunit and a betagamma dimer. Whole cell studies have reported that receptors signal through specific betagamma subtypes. Membrane reconstitution studies with the adenosine A(1) and alpha(2A) adrenergic receptors have reached a similar conclusion. We aimed to test the generality of this finding by comparing the gamma subtype specificity for four G(i)-coupled receptors: alpha(2A) adrenergic; A1 adenosine (A(1)-R); 5-hydroxytryptamine(1A) (5-HT(1A)-R); mu opioid. Membranes were reconstituted with Galpha(i)(1) and five gamma subtypes (dimerized to beta1). Using a sensitive alpha-betagamma binding assay, we show that all recombinant betagamma (except beta1gamma1) had comparable affinity for alpha(i)(1). Using high affinity agonist binding as a measure of receptor-G protein coupling, betagamma-containing gamma11 was the most potent for A(1)-R and 5-HT(1A)-R (p < 0.05, one way ANOVA) while gamma7 was most potent for the other two receptors. gamma11 was 3-8-fold more potent for the A(1)-R than were the other gamma subtypes. Also, gamma11 was 2-8-fold more potent for A(1)-R than at the other receptors, suggesting a unique coupling specificity of the A(1)-R for gamma11. In contrast, the discrimination by receptors for the other betagamma subtypes (beta1 and gamma1, gamma2, gamma7, and gamma10) was limited (2-3-fold). Thus the exquisite betagamma specificity of individual receptors reported in whole cell studies may depend on in vivo mechanisms beyond direct receptor recognition of betagamma subtypes.

The G protein beta subunit is a determinant in the coupling of Gs to the beta 1-adrenergic and A2a adenosine receptors.

The signaling specificity of five purified G protein betagamma dimers, beta(1)gamma(2), beta(2)gamma(2), beta(3)gamma(2), beta(4)gamma(2), and beta(5)gamma(2), was explored by reconstituting them with G(s) alpha and receptors or effectors in the adenylyl cyclase cascade. The ability of the five betagamma dimers to support receptor-alpha-betagamma interactions was examined using membranes expressing the beta(1)-adrenergic or A2a adenosine receptors. These receptors discriminated among the defined heterotrimers based solely on the beta isoform. The beta(4)gamma(2) dimer demonstrated the highest coupling efficiency to either receptor. The beta(5)gamma(2) dimer coupled poorly to each receptor, with EC(50) values 40-200-fold higher than those observed with beta(4)gamma(2). Strikingly, whereas the EC(50) of the beta(1)gamma(2) dimer at the beta(1)-adrenergic receptor was similar to beta(4)gamma(2), its EC(50) was 20-fold higher at the A2a adenosine receptor. Inhibition of adenylyl cyclase type I (AC1) and stimulation of type II (AC2) by the betagamma dimers were measured. betagamma dimers containing Gbeta(1-4) were able to stimulate AC2 similarly, and beta(5)gamma(2) was much less potent. beta(1)gamma(2), beta(2)gamma(2), and beta(4)gamma(2) inhibited AC1 equally; beta(3)gamma(2) was 10-fold less effective, and beta(5)gamma(2) had no effect. These data argue that the beta isoform in the betagamma dimer can determine the specificity of signaling at both receptors and effectors.

Synthesis and structural characterization of a silver complex of a mixed-donor N-heterocyclic carbene linked cyclophane.

The synthesis of a dicationic imidazolium-linked cyclophane and a dimeric silver-N-heterocyclic carbene complex, that is the first silver complex with a N-heterocyclic carbene ligand involved in a pi-bonding interaction, is reported.

Role of C-terminal domains of the G protein beta subunit in the activation of effectors.

The prenyl group on the G protein gamma subunit is an important determinant of protein-protein interactions between the betagamma dimer and its targets, such as alpha subunits, receptors, and effectors. In an effort to identify domains of the beta subunit important for the activation of effectors, we have prepared two types of mutants, one set in the domain suggested to form a hydrophobic prenyl-binding pocket for the gamma subunit's prenyl group (prenyl pocket mutants) and the other set in a domain between Gly(306) and Gly(319) in the beta propeller, which undergoes a conformational change when the dimer binds to phosducin (conformational change mutants). Recombinant baculoviruses for each set of mutants were prepared, and the nine mutant beta subunits were overexpressed with either the gamma(2) subunit (modified with geranylgeranyl) or the gamma(2-L71S) subunit (gamma(2) with altered CAAX sequence and modified with farnesyl). The purified dimers were tested for their ability to couple Galpha(i1) to the A1 adenosine receptor and to activate phospholipase C-beta or type II adenylyl cyclase. All dimers containing mutant beta subunits were indistinguishable from wild-type beta(1)gamma(2) or beta(1)gamma(2-L71S) in coupling the receptor to Galpha(i1). The prenyl pocket mutants expressed with gamma(2) were 10-fold less potent in activating phospholipase C-beta and adenylyl cyclase than beta(1)gamma(2) and had similar activities to beta(1)gamma(2-L71S). The conformational change mutants caused a 15- to 23-fold decrease in EC(50) values for activation of these two effectors. Overall, the results suggest that the sites in Gbeta identified by these mutants are very important in the activation of effectors. Furthermore, the nature of the prenyl group on Ggamma may play an important role in the conformational change leading to the activity of Gbetagamma on effectors.

Activation and inhibition of G protein-coupled inwardly rectifying potassium (Kir3) channels by G protein beta gamma subunits.

G protein-coupled inwardly rectifying potassium (GIRK) channels can be activated or inhibited by different classes of receptors, suggesting a role for G proteins in determining signaling specificity. Because G protein betagamma subunits containing either beta1 or beta2 with multiple Ggamma subunits activate GIRK channels, we hypothesized that specificity might be imparted by beta3, beta4, or beta5 subunits. We used a transfection assay in cell lines expressing GIRK channels to examine effects of dimers containing these Gbeta subunits. Inwardly rectifying K(+) currents were increased in cells expressing beta3 or beta4, with either gamma2 or gamma11. Purified, recombinant beta3gamma2 and beta4gamma2 bound directly to glutathione-S-transferase fusion proteins containing N- or C-terminal cytoplasmic domains of GIRK1 and GIRK4, indicating that beta3 and beta4, like beta1, form dimers that bind to and activate GIRK channels. By contrast, beta5-containing dimers inhibited GIRK channel currents. This inhibitory effect was obtained with either beta5gamma2 or beta5gamma11, was observed with either GIRK1,4 or GIRK1,2 channels, and was evident in the context of either basal or agonist-induced currents, both of which were mediated by endogenous Gbetagamma subunits. In cotransfection assays, beta5gamma2 suppressed beta1gamma2-activated GIRK currents in a dose-dependent manner consistent with competitive inhibition. Moreover, we found that beta5gamma2 could bind to the same GIRK channel cytoplasmic domains as other, activating Gbetagamma subunits. Thus, beta5-containing dimers inhibit Gbetagamma-stimulated GIRK channels, perhaps by directly binding to the channels. This suggests that beta5-containing dimers could act as competitive antagonists of other Gbetagamma dimers on GIRK channels.

Selective coupling of G protein beta gamma complexes to inhibition of Ca2+ channels.

Several mechanisms couple heterotrimeric guanine nucleotide-binding proteins (G proteins) to cellular effectors. Although alpha subunits of G proteins (Galpha) were the first recognized mediators of receptor-effector coupling, Gbetagamma regulation of effectors is now well known. Five Gbeta and 12 Ggamma subunit genes have been identified, suggesting through their diversity that specific subunits couple selectively to effectors. The molecular determinants of Gbetagamma-effector coupling, however, are not well understood, and most studies of G protein-effector coupling do not support selectivity of Gbetagamma action. To explore this issue further, we have introduced recombinant Gbetagamma complexes into avian sensory neurons and measured the inhibition of Ca(2+) currents mediated by an endogenous phospholipase Cbeta- (PLCbeta) and protein kinase C-dependent pathway. Activities of Gbetagamma in the native cells were compared with enzyme assays performed in vitro. We report a surprising selective activation of the PLCbeta pathway by Gbetagamma complexes containing beta(1) subunits, whereas beta(2)-containing complexes produced no activation. In contrast, when assayed in vitro, PLCbeta and type II adenylyl cyclase did not discriminate among these same Gbetagamma complexes, suggesting the possibility that additional cellular determinants confer specificity in vivo.

Role of isoprenoid lipids on the heterotrimeric G protein gamma subunit in determining effector activation.

Post-translational prenylation of heterotrimeric G protein gamma subunits is essential for high affinity alpha-beta gamma and alpha-beta gamma-receptor interactions, suggesting that the prenyl group is an important domain in the beta gamma dimer. To determine the role of the prenyl modification in the interaction of beta gamma dimers with effectors, the CAAX (where A indicates alipathic amino acid) motifs in the gamma1, gamma2, and gamma11 subunits were altered to direct modification with different prenyl groups. Six recombinant beta gamma dimers were overexpressed in baculovirus-infected Sf9 insect cells, purified, and examined for their ability to stimulate three phospholipase C-beta isozymes and type II adenylyl cyclase. The native beta1 gamma2 dimer (gamma subunit modified with geranylgeranyl) is more potent and effective in activating phospholipase C-beta than either the beta1 gamma1 (farnesyl) or the beta1 gamma11 (farnesyl) dimers. However, farnesyl modification of the gamma subunit in the beta1 gamma2 dimer (beta1 gamma2-L71S) caused a decrement in its ability to activate phospholipase C-beta. In contrast, both the beta1 gamma1-S74L (geranylgeranyl) and the beta1 gamma11-S73L (geranylgeranyl) dimers were more active than the native forms. The beta1 gamma2 dimer activates type II adenylyl cyclase about 12-fold; however, neither the beta1 gamma1 nor the beta1 gamma11 dimers activate the enzyme. As was the case with phospholipase C-beta, the beta1gamma2-L71S dimer was less able to activate adenylyl cyclase than the native beta1 gamma2 dimer. Interestingly, neither the beta1 gamma1-S74L nor the beta1 gamma11-S73L dimers stimulated adenylyl cyclase. The results suggest that both the amino acid sequence of the gamma subunit and its prenyl group play a role in determining the activity of the beta gamma-effector complex.

Development of an assay for phospholipase C using column-reconstituted, extruded phospholipid vesicles.

The reconstitution of heterotrimeric G proteins into phospholipid vesicles has been widely used for the measurement of PLC-beta activity in vitro. We have developed an improved and sensitive method for the assay of PLC-beta activity. This approach involves reconstitution of purified betagamma dimers into extruded phospholipid vesicles containing phosphatidylinositol 4, 5-bisphosphate and using a gel-filtration technique to separate the reconstituted vesicles from monodispersed betagamma dimers and the detergent used to solubilize G proteins. The method provides physical information about the partitioning of betagamma dimers into phospholipid vesicles and was used to examine the effect of different prenyl groups on the gamma subunits in the activation of PLC-beta. The beta1gamma1 dimer (containing the farnesyl group) and the beta1gamma2 dimer (containing the geranylgeranyl group) were purified from baculovirus-infected Sf9 insect cells and were found to partition equally into phospholipid vesicles. The beta1gamma2 dimer is more potent and effective in stimulating PLC-beta activity than the beta1gamma1 dimer. The EC50 values of betagamma dimers for the activation of PLC-beta determined with this method were lower than those determined by previous methodology, showing that betagamma subunits have a subnanomolar affinity for PLC-beta.

Instability of the G-protein beta5 subunit in detergent.

Heterotrimeric guanine nucleotide-binding proteins are important mediators in signal transduction and function by transmitting information from membrane-bound receptors to effectors. Because these proteins are membrane bound and contain covalent lipid modifications, detergents are required for solubilization and purification. It was discovered that the interaction between the beta5 subunit and the gamma2 subunit was disrupted in two detergents, cholate and Chaps (3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate). A beta5gamma2 column was constructed in which recombinant betagamma dimers were immobilized on a FLAG antibody column via a hexahistidine-FLAG-tagged gamma2 subunit, gamma2HF. Greater than 95% of the beta5 subunit was specifically eluted from the immobilized gamma2HF subunit using a cholate gradient from 0.05 to 1.0% and greater than 40% of the beta5 subunit was eluted using a Chaps gradient from 0.05 to 1.0%. In contrast, the beta1, beta2, and beta3 subunits remained bound to the gamma2HF subunit in all concentrations of Chaps and cholate. Genapol C-100, Triton X-100, and polyoxyethylene-10-lauryl ether did not interfere with any of the four beta subunits' ability to interact with the gamma2 subunit. These data suggest that the beta5 subunit is not stable in bile acid or Chaps-type detergents (i.e., Chapso, glycocholate, deoxycholate). Therefore, alternative detergents should be used to extract dimers containing the beta5 subunit.

Facilitation of signal onset and termination by adenylyl cyclase.

The alpha subunit (Gsalpha) of the stimulatory heterotrimeric guanosine triphosphate binding protein (G protein) Gs activates all isoforms of mammalian adenylyl cyclase. Adenylyl cyclase (Type V) and its subdomains, which interact with Gsalpha, promoted inactivation of the G protein by increasing its guanosine triphosphatase (GTPase) activity. Adenylyl cyclase and its subdomains also augmented the receptor-mediated activation of heterotrimeric Gs and thereby facilitated the rapid onset of signaling. These findings demonstrate that adenylyl cyclase functions as a GTPase activating protein (GAP) for the monomeric Gsalpha and enhances the GTP/GDP exchange factor (GEF) activity of receptors.

Differential activity of the G protein beta5 gamma2 subunit at receptors and effectors.

The G protein beta5 subunit differs substantially in amino acid sequence from the other known beta subunits suggesting that beta gamma dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the beta5 subunit, recombinant beta5 gamma2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the gamma2 subunit (gamma2HF). The function of the pure beta5 gamma2HF dimers was examined in three assays: activation of pure phospholipase C-beta in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of alpha subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the beta5 gamma2HF dimer was compared with that of the beta1 gamma2HF dimer, which has demonstrated activity in these assays. The beta5 gamma2HF dimer activated phospholipase C-beta with a potency and efficacy similar to that of beta1 gamma2 or beta1 gamma2HF; however, it was markedly less effective than the beta1 gamma2HF or beta1 gamma2 dimer in its ability to activate type II adenylyl cyclase (EC50 of approximately 700 nM versus 25 nM). Both the beta5 gamma2HF and the beta1 gamma2HF dimers supported coupling of M1 muscarinic receptors to the Gq alpha subunit. The ETB receptor coupled effectively to both the Gi and Gq alpha subunits in the presence of the beta1 gamma2HF dimer. In contrast, the beta5 gamma2HF dimer only supported coupling of the Gq alpha subunits to the ETB receptor and did not support coupling of the Gi alpha subunit. These results suggest that the beta5 gamma2HF dimer binds selectively to Gq alpha subunits and does not activate the same set of effectors as dimers containing the beta1 subunit. Overall, the data support a specialized role for the beta5 subunit in cell signaling.

Phosphorylation of the G protein gamma12 subunit regulates effector specificity.

Although the G protein betagamma dimer is an important mediator in cell signaling, the mechanisms regulating its activity have not been widely investigated. The gamma12 subunit is a known substrate for protein kinase C, suggesting phosphorylation as a potential regulatory mechanism. Therefore, recombinant beta1 gamma12 dimers were overexpressed using the baculovirus/Sf9 insect cell system, purified, and phosphorylated stoichiometrically with protein kinase C alpha. Their ability to support coupling of the Gi1 alpha subunit to the A1 adenosine receptor and to activate type II adenylyl cyclase or phospholipase C-beta was examined. Phosphorylation of the beta1 gamma12 dimer increased its potency in the receptor coupling assay from 6.4 to 1 nM, changed the Kact for stimulation of type II adenylyl cyclase from 14 to 37 nM, and decreased its maximal efficacy by 50%. In contrast, phosphorylation of the dimer had no effect on its ability to activate phospholipase C-beta. The native beta1gamma10 dimer, which has 4 similar amino acids in the phosphorylation site at the N terminus, was not phosphorylated by protein kinase C alpha. Creation of a phosphorylation site in the N terminus of the protein (Gly4 --> Lys) resulted in a beta1 gamma10G4K dimer which could be phosphorylated. The activities of this beta gamma dimer were similar to those of the phosphorylated beta1 gamma12 dimer. Thus, phosphorylation of the beta1 gamma12 dimer on the gamma subunit with protein kinase C alpha regulates its activity in an effector-specific fashion. Because the gamma12 subunit is widely expressed, phosphorylation may be an important mechanism for integration of the multiple signals generated by receptor activation.

The G protein beta5 subunit interacts selectively with the Gq alpha subunit.

The diversity in the heterotrimeric G protein alpha, beta, and gamma subunits may allow selective protein-protein interactions and provide specificity for signaling pathways. We examined the ability of five alpha subunits (alphai1, alphai2, alphao, alphas, and alphaq) to associate with three beta subunits (beta1, beta2, and beta5) dimerized to a gamma2 subunit containing an amino-terminal hexahistidine-FLAG affinity tag (gamma2HF). Sf9 insect cells were used to overexpress the recombinant proteins. The hexahistidine-FLAG sequence does not hinder the function of the beta1gamma2HF dimer as it can be specifically eluted from an alphai1-agarose column with GDP and AlF4-, and purified beta1gamma2HF dimer stimulates type II adenylyl cyclase. The beta1gamma2HF and beta2gamma2HF dimers immobilized on an anti-FLAG affinity column bound all five alpha subunits tested, whereas the beta5gamma2HF dimer bound only alphaq. The ability of other alpha subunits to compete with the alphaq subunit for binding to the beta5gamma2HF dimer was tested. Addition of increasing amounts of purified, recombinant alphai1 to the alphaq in a Sf9 cell extract did not decrease the amount of alphaq bound to the beta5gamma2HF column. When G proteins in an extract of brain membranes were activated with GDP and AlF4- and deactivated in the presence of equal amounts of the beta1gamma2HF or beta5gamma2HF dimers, only alphaq bound to the beta5gamma2HF dimer. The alphaq-beta5gamma2HF interaction on the column was functional as GDP, and AlF4- specifically eluted alphaq from the column. These results indicate that although the beta1 and beta2 subunits interact with alpha subunits from the alphai, alphas, and alphaq families, the structurally divergent beta5 subunit only interacts with alphaq.

Expression, purification, and regulation of two isoforms of the inositol 1,4,5-trisphosphate 3-kinase.

The level of inositol 1,4,5-trisphosphate in the cytoplasm is tightly regulated by two enzymes, the inositol 1,4,5,5-phosphatase and the inositol 1,4,5-trisphosphate 3-kinase. Two isoforms of the inositol 1,4,5-trisphosphate 3-kinase have been identified, the A form and the B form. The regulatory properties of the two isoforms were compared following overexpression and purification of the proteins from a v-src transformed mammalian cell line. The highly purified, recombinant inositol 1,4,5-trisphosphate 3-kinases were differentially regulated by calcium/calmodulin and via phosphorylation by protein kinase C or the cyclic AMP-dependent protein kinase. Both enzymes had similar affinities for inositol 1,4, 5-trisphosphate (Km 2-5 mu M). Calcium/calmodulin stimulated the activity of isoform A about 2.5-fold, whereas the activity of isoform B was increased 20-fold. The cyclic AMP-dependent protein kinase phosphorylated the inositol 1,4,5-trisphosphate 3-kinase A to the extent of 0.9 mol/mol and isoform B to 1 mol/mol. Protein kinase C phosphorylated isoform A to the extent of 2 mol/mol and isoform B to 2.7 mol/mol. Phosphorylation of isoform A by the cyclic AMP-dependent protein kinase caused a 2.5-fold increase in its activity when assayed in the absence of calcium/calmodulin, whereas phosphorylation by protein kinase C decreased activity by 72%. The activity of isoform B in the absence of calcium/calmodulin was not affected by phosphorylation using either kinase. When assayed in the presence of calcium/calmodulin, phosphorylation of isoform A by the cyclic AMP-dependent protein kinase increased activity 1.5-fold, whereas phosphorylation of isoform B decreased activity by 45%. Phosphorylation of either isoform A or B by protein kinase C resulted in a 70% reduction of calcium/calmodulin-stimulated activity. Differential expression and regulation of the two inositol 1,4,5-trisphosphate 3-kinase isoforms provides multiple mechanisms for regulating the cytosolic level of inositol 1,4,5-trisphosphate in cells.

Activation of the calcium-permeable cation channel CD20 by alpha subunits of the Gi protein.

When the calcium-permeable cation channel CD20 is expressed in Balb/c 3T3 cells, it is activated by insulin-like growth factor-I (IGF-I) via the IGF-I receptor (Kanzaki, M., Nie, L., Shibata, H., and Kojima, I. (1997) J. Biol. Chem. 272, 4964-4969). The present study was conducted to investigate the role of G proteins in the regulation of the CD20 channel. In the excised patch clamp mode, activation of the CD20 channel by IGF-I required GTP, Mg2+, and ATP in the bath solution, and removal of either GTP or ATP attenuated the activation. Non-hydrolyzable ATP could substitute for ATP, and guanyl-5'-yl thiophosphate blocked the activation of the channel by IGF-I. The CD20 channel was also activated by guanosine 5'-3-O-(thio)triphosphate, and ATP was not required for the activation. Addition of a preparation of Gi/Go holoprotein purified from bovine brain activated the CD20, and the beta-adrenergic receptor kinase peptide did not affect the number of channel openings induced by the G protein. The CD20 channel was stimulated by the GTP-bound form of recombinant Gi2 alpha subunit purified from Sf9 cells. The Gi3 alpha subunit was less effective, and the Gi1 alpha subunit had no effect. Purified recombinant beta1gamma2 subunits did not affect the activity of the channel. Finally, IGF-I-induced activation of CD20 was inhibited by an antibody against Gi2 alpha subunit. These findings indicate that the CD20 channel expressed in Balb/c 3T3 cells is activated by the IGF-I receptor via the alpha subunits of heterotrimeric G proteins.

Tubulin, Gq, and phosphatidylinositol 4,5-bisphosphate interact to regulate phospholipase Cbeta1 signaling.

The cytoskeletal protein, tubulin, has been shown to regulate adenylyl cyclase activity through its interaction with the specific G protein alpha subunits, Galphas or Galphai1. Tubulin activates these G proteins by transferring GTP and stabilizing the active nucleotide-bound Galpha conformation. To study the possibility of tubulin involvement in Galphaq-mediated phospholipase Cbeta1 (PLCbeta1) signaling, the m1 muscarinic receptor, Galphaq, and PLCbeta1 were expressed in Sf9 cells. A unique ability of tubulin to regulate PLCbeta1 was observed. Low concentrations of tubulin, with guanine nucleotide bound, activated PLCbeta1, whereas higher concentrations inhibited the enzyme. Interaction of tubulin with both Galphaq and PLCbeta1, accompanied by guanine nucleotide transfer from tubulin to Galphaq, is suggested as a mechanism for the enzyme activation. The PLCbeta1 substrate, phosphatidylinositol 4,5-bisphosphate, bound to tubulin and prevented microtubule assembly. This observation suggested a mechanism for the inhibition of PLCbeta1 by tubulin, since high tubulin concentrations might prevent the access of PLCbeta1 to its substrate. Activation of m1 muscarinic receptors by carbachol relaxed this inhibition, probably by increasing the affinity of Galphaq for tubulin. Involvement of tubulin in the articulation between PLCbeta1 signaling and microtubule assembly might prove important for the intracellular governing of a broad range of cellular events.

Reconstitution of bovine A1 adenosine receptors and G proteins in phospholipid vesicles: betagamma-subunit composition influences guanine nucleotide exchange and agonist binding.

We have studied the interactions of purified A1 adenosine receptors and G proteins reconstituted into phospholipid vesicles to investigate how the betagamma composition of G protein heterotrimers influences coupling. Recombinant hexahistidine-tagged bovine A1 adenosine receptors were expressed in Sf9 cells and purified to homogeneity by sequential chromatography over heparin-sepharose, xanthine amino congener-agarose, and nickel-nitrilotriacetic acid columns. These receptors were reconstituted with pure recombinant G proteins of defined subunit composition. Receptor-G protein complexes containing alphai2 and beta1gamma2 or beta1gamma3 and stimulated with the agonist, (R)-phenylisopropyladenosine, exchange guanine nucleotide 2-3 times more rapidly than do complexes containing beta1gamma1. This difference is not overcome by increasing the concentration of betagamma subunits. Receptor-G protein complexes containing beta1gamma1 also bind less of the agonist, [125I]-iodoaminobenzyladenosine (125I-ABA), than do complexes containing beta1gamma3. Kinetic experiments show that 125I-ABA dissociates 2-fold more rapidly from receptor-G protein complexes containing beta1gamma1 than from complexes containing the other betagamma subunits. The affinity of the interaction between immobilized Galphai2 subunits and beta1gamma1 or beta1gamma2 measured with an optical biosensor in the absence of receptor is similar. Taken together, these data implicate the gamma-subunit in influencing the interaction between the A1 adenosine receptor and G proteins.

Reconstitution of recombinant bovine A1 adenosine receptors in Sf9 cell membranes with recombinant G proteins of defined composition.

We investigated the coupling of A1 adenosine receptors to recombinant G proteins. Recombinant baculoviruses were used to express bovine A1 adenosine receptors in Sf9 insect cells that lack endogenous adenosine receptors. Binding parameters for recombinant receptors expressed in Sf9 cell membranes using the antagonist radioligand [125I]BW-A844U ([125I]8-cyclopentyl-3-iodoaminophenethyl-1-propylxanthine) are Bmax = 2-5 pmol/mg of protein and K(D) = 0.53 +/- 0.12 nM. In competition assays, the potency order of agonists is (R)-phenylisopropyladenosine > (S)-phenylisopropyladenosine > 5'-N-ethylcarboxamidoadenosine, properties characteristic of native bovine A1 adenosine receptors. The agonist radioligand 125I-N6-4-aminobenzyladenosine binds to two affinity states of the recombinant A1 adenosine receptors with K(D) values of 0.09 and 10.4 nM. The high affinity binding site represents <10% of total sites and is increased 7-fold on reconstitution with both alpha and betagamma G protein subunits but not with either subunit alone; thus, exogenous alpha and betagamma subunits do not functionally interact with endogenous Sf9 betagamma and alpha subunits, respectively. Four different alpha subunits (alpha i1, alpha i2, alpha i3, and alpha o) and six different beta gamma subunits (beta1gamma1, beta1gamma2, beta1gamma3, beta2gamma2, beta2gamma3, and bovine brain betagamma)) increased GTP-sensitive, high affinity agonist binding. The results indicate that bovine A1 adenosine receptors couple equally well to G protein alpha i and alpha o subunits in combination with betagamma subunits containing the beta1 or beta2 subunits and gamma2 or gamma3 subunits. G protein heterotrimers that contain the beta1gamma1 dimer couple with similar potency but reduced efficacy to A1 adenosine receptors.

Transformation of Rat-1 fibroblasts with the v-src oncogene induces inositol 1,4,5-trisphosphate 3-kinase expression.

Transformation of Rat-1 fibroblasts with the v-src oncogene leads to a 6- to 8-fold enhancement of the activity of the Ins(1,4,5)P3 3-kinase in cytosolic extracts [Johnson, Wasilenko, Mattingly, Weber and Garrison (1989) Science 246, 121-124]. This study confirms these results using another v-src-transformed Rat-1 cell line (B31 cells) and investigates the molecular mechanism by which pp60v-src activates Ins(1,4,5)P3 3-kinase. The mRNA and protein levels for two rat isoforms of Ins(1,4,5)P3 3-kinase were determined in the v-src-transformed cell line. Both the mRNA and protein levels for isoform A were elevated in v-src-transformed Rat-1 cells while those for isoform B were not significantly affected. Moreover, stable expression of either form of Ins(1,4,5)P3 3-kinase in the B31 v-src-transformed Rat-1 cell line did not result in tyrosine phosphorylation of Ins(1,4,5)P3 3-kinase A or B. These results suggest that at least one mechanism by which the v-src oncogene increases the activity of the Ins(1,4,5)P3 3-kinase in the Rat-1 transformed fibroblast is by increasing the level of expression of Ins(1,4,5)P3 3-kinase A.