The long term objective of this research project is to develop a detailed molecular understanding of signaling through G protein pathways. Many hormones, auto and paracrine factors regulate cell function by binding to receptors that use heterotrimeric G proteins as signal transducers. Hormone occupied receptors activate G proteins and promote the dissociation of the GTP bound alpha-subunit from beta gamma-subunits. Both the free alpha and beta gamma subunits regulate the activities of effectors such as adenylyl cyclase(AC), phospholipase C(PLC) and K+ channels. While all isoforms of adenylyl cyclase and phospholipase C are stimulated by alpha-subunits, only some forms such as AC1, 2 and 4 and PLC-beta2 and beta 3 are regulated by beta gamma-subunits. In the AC system AC2 and 4 are conditionally stimulated by beta gamma-subunits while AC1 is inhibited. These observations suggest that signal transmission by beta gamma-subunits may play an important role in integrated biological response. Regulation of effectors by G protein subunits occur by non-covalent protein-protein interactions. Up to now there has been no information about the regions involved or interactions that underlie the regulation of effectors by beta gamma-subunits. We have recently identified the region between aa 956-982 of AC2 as being involved in receiving signals from beta gamma-subunits. A peptide (called QEHA) encoding this region specifically blocks beta gamma but not alpha/s or forskolin stimulation of AC2. The QEHA peptide also blocks beta gamma inhibition of AC1. In addition the QEHA peptide specifically blocks beta gamma stimulation of PLC-beta3, K channels and beta-adrenergic receptor kinase but does not affect beta gamma interactions with alpha-subunits. The experiments for the current term build on this finding and will focus on beta gamma-regulation of effectors with special emphasis on AC2 and AC1. We will characterize in detail the region of AC2 involved interaction with beta gamma-subunits. For this we will use purified epitope tagged AC2. We will crosslink beta gamma to AC2 and demonstrate that the QEHA peptide can specifically block the crosslinking. We will synthesize additional peptides to map the minimum regions within aa 956- 982 AC2 required for interaction with beta gamma subunits. We will determine the key residues in AC2 that involved in this interaction by synthesis of Ala substituted peptides as well point mutations on AC2. We will use the QEHA peptide to identify and characterize the regions of beta-subunits involved in interaction with effectors. For this we will use recombinant beta2gamma2 expressed in Sf9 cells. We will crosslink the QEHA peptide to the beta-subunit, digest the beta-subunit and identify the fragment that has the crosslink by HPLC and mass spectrometry. From the sequence information obtained from the mass spectrometry experiments we will synthesize peptides encoding sequences of beta-subunits and test if the peptides block beta gamma-regulation of effectors. To identify key residues on the beta-subunit, we will synthesize Ala substituted peptides and score for loss of activity of the modified peptides. We will also mutate individual key residues on beta-subunits and determine if mutations result in loss of beta gamma regulation of effector. We will identify and characterize the regions of AC1 that are involved in beta gamma-interactions. For this we will use approaches similar to that described above for AC2. From these experiments we will obtain a clear understanding of the residues and hence the interactions involved in beta gamma regulation of effectors. From the comparative studies on AC1 and AC2 we will gain an understanding of the interactions that need to be conserved so that beta gamma subunits can regulate the activity of effectors. Such understanding could lead to new long acting therapeutic agents for many diseases.
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| Korgaonkar, Sonal Navin; Feng, Xiaobei; Ross, Michael D et al. (2008) HIV-1 upregulates VEGF in podocytes. J Am Soc Nephrol 19:877-83 |
| Neves, Susana R; Tsokas, Panayiotis; Sarkar, Anamika et al. (2008) Cell shape and negative links in regulatory motifs together control spatial information flow in signaling networks. Cell 133:666-80 |
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