Distribution and relative levels of expression of the phosphoinositidase-C-linked G-proteins Gq alpha and G11 alpha: absence of G11 alpha in human platelets and haemopoietically derived cell lines.

Membranes of a variety of clonal cell lines, including neuroblastoma x glioma hybrid NG108-15, glioma C6, Rat 1 and CHO fibroblasts and the pituitary-derived cell lines alpha T3 and GH3 were immunoblotted with an antiserum (CQ) raised against a synthetic peptide corresponding to the C-terminal decapeptide of the alpha subunits of the phosphoinositidase-C-linked G-proteins Gq and G11. In SDS-PAGE conditions able to resolve these two polypeptides, direct evidence was obtained for co-expression of these two G-proteins in all of the above cell lines. The ratio of these two G-proteins varied substantially (alpha 11/alpha q = 0.25-2.5) between the cell lines. In human platelets and in a range of haemopoietically derived human cell lines including U937 (monoblasts), Raji (Burkitts lymphoma) and Jurkat (mature T cell) expression of G11 alpha was not detected. This was not due to the inability of the antiserum to identify human G11 alpha as other human cell lines co-expressed both G-proteins. A third, unidentified CQ reactive polypeptide of similar mobility was resolved and present in all cell lines examined.

The human muscarinic M1 acetylcholine receptor, when express in CHO cells, activates and downregulates both Gq alpha and G11 alpha equally and non-selectively.

CHO cells express both of the phosphoinositidase C-linked G-proteins Gq and G11. G11 alpha is some 2.5-fold more highly expressed than Gq alpha in membranes of these cells. Following transfection and stable expression of CHO cells with DNA encoding the human muscarinic M1 acetylcholine (HM1) receptor, chronic treatment of the cells with the cholinergic agonist carbachol resulted in down-regulation of membrane levels of both Gq alpha and G11 alpha. Dose-response curves to carbachol produced identical EC50 values for agonist-induced down-regulation of the two G-proteins and both were down-regulated with the same time course. These data indicate that the HM1 receptor interacts with the activates both Gq alpha and G11 alpha equivalently and non-selectively in a whole cell system in which the receptor has access to both G-proteins.

cAMP-specific phosphodiesterase HSPDE4D3 mutants which mimic activation and changes in rolipram inhibition triggered by protein kinase A phosphorylation of Ser-54: generation of a molecular model.

Ser-13 and Ser-54 were shown to provide the sole sites for the protein kinase A (PKA)-mediated phosphorylation of the human cAMP-specific phosphodiesterase isoform HSPDE4D3. The ability of PKA to phosphorylate and activate HSPDE4D3 was mimicked by replacing Ser-54 with either of the negatively charged amino acids, aspartate or glutamate, within the consensus motif of RRES54. The PDE4 selective inhibitor rolipram ¿4-[3-(cyclopentoxy)-4-methoxyphenyl]-2-pyrrolidone¿ inhibited both PKA-phosphorylated HSPDE4D3 and the Ser-54-->Asp mutant, with an IC50 value that was approximately 8-fold lower than that seen for the non-PKA-phosphorylated enzyme. Lower IC50 values for inhibition by rolipram were seen for a wide range of non-activated residue 54 mutants, except for those which had side-chains able to serve as hydrogen-bond donors, namely the Ser-54-->Thr, Ser-54-->Tyr and Ser-54-->Cys mutants. The Glu-53-->Ala mutant exhibited an activity comparable with that of the PKA phosphorylated native enzyme and the Ser-54-->Asp mutant but, in contrast to the native enzyme, was insensitive to activation by PKA, despite being more rapidly phosphorylated by this protein kinase. The activated Glu-53-->Ala mutant exhibited a sensitivity to inhibition by rolipram which was unchanged from that of the native enzyme. The double mutant, Arg-51-->Ala/Arg-52-->Ala, showed no change in either enzyme activity or rolipram inhibition from the native enzyme and was incapable of providing a substrate for PKA phosphorylation at Ser-54. No difference in inhibition by dipyridamole was seen for the native enzyme and the Ser-54-->Asp and Ser-54-->Ala mutants. A model is proposed which envisages that phosphorylation by PKA triggers at least two distinct conformational changes in HSPDE4D3; one of these gives rise to enzyme activation and another enhances sensitivity to inhibition by rolipram. Activation of HSPDE4D3 by PKA-mediated phosphorylation is suggested to involve disruption of an ion-pair interaction involving the negatively charged Glu-53. The increase in susceptibility to inhibition by rolipram upon PKA-mediated phosphorylation is suggested to involve the disruption of a hydrogen-bond involving the side-chain hydroxy group of Ser-54.

The palmitoylation status of the G-protein G(o)1 alpha regulates its activity of interaction with the plasma membrane.

Plasmids containing cDNAs encoding either the wild-type guanine-nucleotide-binding protein G(o)1 alpha or the palmitoylation-negative cysteine-3-to-serine (C3S) mutant of G(o)1 alpha were transfected into Rat 1 cells, and clones stably expressing immunoreactivity corresponding to these polypeptides were isolated. Clones C5B (expressing wild-type G(o)1 alpha) and D3 (expressing the mutant form) were selected for detailed study. Immunoprecipitation of whole cell lysates of each clone labelled with either [3H]palmitate or [3H]myristate demonstrated incorporation of [3H]myristate into both wild-type and the C3S mutant of G(o)1 alpha, but that incorporation of hydroxylamine-sensitive [3H]palmitate was restricted to the wild type. When membrane and cytoplasmic fractions were prepared from cells of either the C5B or D3 clones, although immunodetection of wild-type G(o)1 alpha was observed only in the membrane fraction, the C3S mutant was present in both membrane and cytoplasmic fractions. Furthermore, a significant proportion of the C3S G(o)1 alpha immunoreactivity was also detected in the cytoplasmic fraction if immunoprecipitation of recently synthesized G(o)1 alpha was performed from fractions derived from cells pulse-labelled with [35S]Trans label. Pretreatment of cells of both clones C5B and D3 with pertussis toxin led to complete ADP-ribosylation of the cellular population of G(o)1 alpha in both cell types, irrespective of whether the polypeptide was subsequently found in the membrane or cytoplasmic fraction following cellular disruption. By contrast, separation of membrane and cytoplasmic fractions before pertussis-toxin-catalysed [32P]ADP-ribosylation allowed modification only of the membrane-associated G(o)1 alpha (whether wild-type or the C3S mutant). This labelling was decreased substantially by incubation of the membranes with guanosine 5'-[beta gamma-imido]triphosphate. No cytoplasmic G-protein beta subunit was detected immunologically, and the non-membrane-associated C3S G(o)1 alpha from D3 cells migrated as an apparently monomeric 40 kDa protein on a Superose 12 gel-filtration column. Membrane-associated wild-type and C3S G(o)1 alpha appeared to interact with guanine nucleotides with similar affinity, as no alteration in the dose-response curves for guanine-nucleotide-induced maintenance of a stable 37 kDa tryptic fragment was noted for the two forms of G(o)1 alpha. Chemical depalmitoylation of membranes of clone C5B with neutral 1 M hydroxylamine caused a release of some 25-30% of each of G(o)1 alpha, Gi2 alpha and Gq alpha/G11 alpha from the membranes. Equivalent treatment of D3 cells caused an equivalent release of Gi2 alpha and Gq alpha/G11 alpha, but was unable to cause any appreciable release of the CS3 form of G(o)1 alpha, which was membrane-bound.

The role of palmitoylation of the guanine nucleotide binding protein G11 alpha in defining interaction with the plasma membrane.

Mutations of Cys-9 to serine, Cys-10 to serine and a combination of both alterations were produced in a cDNA encoding murine G11 alpha to potentially interfere with the ability of the expressed polypeptides to act as substrates for post-translational palmitoylation. Each of these mutants and the wild-type protein were expressed in simian COS-1 cells. Mutation of either cysteine-9 or cysteine-10 decreased the degree of palmitoylation of the protein by some 80% compared with the wild-type, while the double mutant totally failed to incorporate [3H]palmitate. By contrast, in all transfections the endogenously expressed simian G11 alpha incorporated [3H]palmitate to similar levels. Particulate and cytoplasmic fractions from these cells were subjected to SDS/PAGE under conditions which allow resolution of primate and rodent forms of G11 alpha. Immunoblotting of these fractions demonstrated that in all cases the endogenously expressed simian G11 alpha was exclusively associated with the particulate fraction, as was the transfected and expressed wild-type murine G11 alpha. By contrast, each of the mutated forms of murine G11 alpha displayed a distribution in which approx. 70% of the expressed protein was present in the particulate fraction and 30% in the supernatant. To examine the conformation of the particulate expressed forms of murine G11 alpha, these fractions were treated with various concentrations of sodium cholate and immunoblots were subsequently performed on the solubilized and remaining particulate proteins. Whereas essentially all of the endogenous simian G11 alpha was solubilized by treatment with 1% (w/v) sodium cholate and some 50% with 0.32% cholate, expressed wild-type murine G11 alpha was more recalcitrant to solubilization. However, that fraction of wild-type murine G11 alpha which was solubilized behaved identically to the endogenous simian G11 alpha on Superose-12 gel-exclusion chromatography. The particulate fraction of the C9S/C10S double mutant of murine G11 alpha was highly resistant to solubilization by sodium cholate, whereas the particulate fractions of the two single cysteine to serine mutants were intermediate to the wild-type and double mutant in their ability to be solubilized by this detergent. These data demonstrate that the palmitoylation status of the cysteine residues at positions 9 and 10 in murine G11 alpha plays a central role in defining membrane association of this G-protein and indicate that much of the particulate fraction of the expressed palmitoylation-resistant mutants is likely to represent non-functional rather than correctly folded protein.(ABSTRACT TRUNCATED AT 400 WORDS)

The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5).

The PDE4A (type IV) cAMP-specific, rolipram-inhibited phosphodiesterase RPDE-6 (RNPDE4A5), when transiently expressed in COS7 cells, could be complexed with the v-Src-SH3 domain expressed as a glutathione S-transferase (GST) fusion protein. RPDE-6 did not interact with GST itself. This complex was not disrupted by treatment with high NaCl concentration together with Triton X-100. Interaction was apparently determined by the N-terminal splice region of RPDE-6, as the PDE4A splice variant RPDE-39, which differs from RPDE-6 at the extreme N-terminus, failed to associate with v-Src-SH3; met26RD1 (where RD1 is rat 'dunc-like' PDE), which has the N-terminal splice region deleted, failed to associate with v-Src-SH3, and the association of RPDE-6 and v-Src-SH3 was blocked by a fusion protein formed from the N-terminal splice region. RDPE-6 showed binding to GST fusion proteins of both the intact Src kinase and an SH2-SH3 construct but did not bind to the Src-SH2 domain or to the adaptor protein Grb-2. RPDE-6 could be co-immunoprecipitated from cytosol extracts of transfected cells by using anti-Src antiserum. RPDE-6 exhibited selectivity in binding to the SH3 domains of c-Abl, Crk, Csk, Lck, Lyn, Fyn and v-Src, with binding to the SH3 regions of the Src-related tyrosyl kinases Lyn and Fyn being the most effective. The binding of RPDE-6 to the SH3 domains of Crk, Csk and Lck led to a marked reduction in PDE activity, but no change was apparent in complexes with other species. Endogenous RPDE-6 from brain, but not endogenous RPDE-39 from testis, bound to the Src-SH3 domain. We suggest that the PDE4A splice variant RPDE-6 has a propensity for interaction with selective SH3 domains, in particular those from Src and the Src-related tyrosyl kinases Lyn and Fyn. This interaction seems to be governed by alternative splicing of the PDE4A gene, because RPDE-39, a splice variant that lacks the proline-rich N-terminal splice region of RPDE-6, does not interact with these SH3 domains. It is proposed that the binding site on RPDE-6 for SH3 domains lies within the unique first 102 residues of its N-terminal splice domain, where two motifs representing Class I SH3 binding sites with selectivity for Src kinase SH3 domains can be identified and one motif for a putative Class II SH3 binding site.