Effect of inactivating mutations on phosphorylation and internalization of the human VPAC2 receptor

The VPAC2 receptor, as all members of the G-protein-coupled receptor (GPCR)-B family, has two highly conserved motifs in the third intracellular (IC3) loop: a lysine and a leucine located at the amino-terminus and two basic residues separated by a leucine and an alanine at the carboxyl-terminus. This study evaluates the involvement of those conserved amino acid sequences in VPAC2 signal transduction and regulation. The residues were mutated into alanine and mutants were expressed in Chinese hamster ovary (CHO) cells stably transfected with Gα16 and aequorin. Mutation of L310 reduced efficacy of vasoactive intestinal polypeptide (VIP) to stimulate adenylate cyclase activity through Gαs coupling by 75%, without affecting VIP capability to stimulate an increase in [Ca2+]i through Gα16 coupling. Mutation of R325 and, to a lesser extend, K328 reduced VIP efficacy to stimulate [Ca2+]i increase and VIP potency to stimulate adenylate cyclase. The combination of mutations of both amino- and carboxyl-terminus located conserved motifs of the IC3 loop generates an inactive receptor with respect to [Ca2+]i increase and adenylate cyclase activation, but also with respect to receptor phosphorylation and internalization that were indeed directly correlated with the potency of inactivation of the receptors. The amino-terminus of the VPAC2 receptor IC3 loop is thus involved in adenylate cyclase activation and the carboxyl-terminus of the IC3 loop participates in both Gαs and Gα16 coupling. The mutations studied also reduced both receptor phosphorylation and internalization in a manner that appeared directly linked to the alteration of Gαs and Gα16 coupling.

Protein Kinases Associated with Activation of Sickle Red Blood Cell Adhesion.

We have previously found that activation of adenylate cyclase in sickle red cells (SS RBCs) results in increased RBC adhesion to cultured endothelial cells (EC) through activation of protein kinase A (PKA). Protein tyrosine phosphorylation by src family kinases, known to be present in normal RBCs, is also involved in enhancing SS RBC adhesion to EC, as inhibition of these kinases also inhibits activation of adhesion. However, in normal RBCs neither activation of adenylate cyclase nor cAMP analogs elicits a similar increase in cell adhesion. We therefore investigated whether normal and SS RBCs differed with respect to the protein kinases downstream of cAMP, such as PKA catalytic and regulatory subunits and protein tyrosine kinases p56lck, p56lyn and p72syk. We also investigated the extracellular signal regulated kinases p44 and p42 (ERK 1 and 2 respectively), which can be activated downstream of tyrosine kinases. PKA catalytic and regulatory subunits and ERK1/2 were studied after SS and normal RBCs were sham-treated or treated for 30 min with 80 μM forskolin, which activates adenylate cyclase. For protein tyrosine kinases, both SS and normal RBCs were sham-treated or treated with 1μg/ml Pertussis toxin, which suppresses Gαi-mediated inhibition of adenylate cyclase. Equal amounts of total membrane proteins were separated by polyacrylamide gel electrophoresis before transfer to nitrocellulose membrane. Protein kinases were detected in isolated membranes by immunoblotting using specific antibodies. Immunoblots showed that the levels of membrane-associated PKAcβ subunit were similar in both SS and normal RBCs and did not change after forskolin stimulation. However, treatment of SS RBCs with forskolin induced a significant reduction in membrane-bound PKARIIβ subunit. In general, dissociation of PKAR (regulatory) subunits is associated with activation of the catalytic (c) domain. In contrast, no change in the amount of membrane-bound PKARIIβ was observed when normal RBCs were treated with forskolin. These data demonstrate that PKARIIβ dissociated from PKAcβ after adenylate cyclase activation in SS RBCs, consistent with activation of PKAcβ. Our results thus suggest that activation of PKAcβ might be one of the mechanisms contributing to increased SS RBC adhesion. The amount of membrane-bound PKARIIα in SS RBCs did not change with stimulation, suggesting that PKAcα may not undergo activation as a result of forskolin stimulation. Immunoblots also showed no change in the levels of membrane-bound p56lck, p56lyn and p72syk detected before and after normal or SS RBCs were stimulated with Pertussis toxin. However, normal RBCs contained lower levels of protein tyrosine kinases, including p56lyn, p56lck and p76syk. We also found that ERK1/2 are bound to the membrane in normal and SS RBCs. The total amount of membrane-associated ERK1 (p44) is markedly higher in SS RBCs than in normal RBCs, and membrane ERK1 levels increased only in SS RBCs after stimulation with forskolin. Taken together, our results suggest that PKAcβ is activated in SS RBCs but not in normal RBCs in response to elevation of cAMP. The higher levels of src kinases in SS RBCs may also contribute to an increased adhesive response to adenylate cyclase activation compared to normal RBCs. In addition, we have obtained the first evidence that ERK1/2 is conserved in enucleated human erythrocytes and that ERK1 is recruited to bind to SS RBC membranes as a result of adenylate cyclase activation, suggesting that it might be involved in regulating cell adhesion.

Schizosaccharomyces pombe Git7p, a Member of the Saccharomyces cerevisiae Sgt1p Family, Is Required for Glucose and Cyclic AMP Signaling, Cell Wall Integrity, and Septation

ABSTRACT The Schizosaccharomyces pombe fbp1 gene, encoding fructose-1,6-bisphosphatase, is transcriptionally repressed by glucose. Mutations that confer constitutive fbp1 transcription identify git (glucose-insensitive transcription) genes that encode components of a cyclic AMP (cAMP) signaling pathway required for adenylate cyclase activation. Four of these genes encode the three subunits of a heterotrimeric G protein (gpa2, git5, and git11) and a G protein-coupled receptor (git3). Three additional genes, git1, git7, and git10, act in parallel to or downstream from the G protein genes. Here, we describe the cloning and characterization of the git7 gene. The Git7p protein is a member of the Saccharomyces cerevisiae Sgt1p protein family. In budding yeast, Sgt1p associates with Skp1p and plays an essential role in kinetochore assembly, while in Arabidopsis, a pair of SGT1 proteins have been found to be involved in plant disease resistance through an interaction with RAR1. Like S. cerevisiae Sgt1p, Git7p is essential, but this requirement appears to be due to roles in septation and cell wall integrity, which are unrelated to cAMP signaling, as S. pombe cells lacking either adenylate cyclase or protein kinase A are viable. In addition, git7 mutants are sensitive to the microtubule-destabilizing drug benomyl, although they do not display a chromosome stability defect. Two alleles of git7 that are functional for cell growth and septation but defective for glucose-triggered cAMP signaling encode proteins that are altered in the highly conserved carboxy terminus. The S. cerevisiae and human SGT1 genes both suppress git7-93 but not git7-235 for glucose repression of fbp1 transcription and benomyl sensitivity. This allele-specific suppression indicates that the Git7p/Sgt1p proteins may act as multimers, such that Git7-93p but not Git7-235p can deliver the orthologous proteins to species-specific targets. Our studies suggest that members of the Git7p/Sgt1p protein family may play a conserved role in the regulation of adenylate cyclase activation in S. pombe, S. cerevisiae, and humans.