Polarity Exchange at the Interface of Regulators of G Protein Signaling with G Protein α-Subunits

RGS proteins are GTPase-activating proteins (GAPs) for G protein alpha -subunits. This GAP activity is mediated by the interaction of conserved residues on regulator of G protein signaling (RGS) proteins and Galpha -subunits. We mutated the important contact sites Glu-89, Asn-90, and Asn-130 in RGS16 to lysine, aspartate, and alanine, respectively. The interaction of RGS16 and its mutants with Galpha t and Galpha i1 was studied. The GAP activities of RGS16N90D and RGS16N130A were strongly attenuated. RGS16E89K increased GTP hydrolysis of Galpha i1 by a similar extent, but with an about 100-fold reduced affinity compared with non-mutated RGS16. As Glu-89 in RGS16 is interacting with Lys-210 in Galpha i1, this lysine was changed to glutamate for compensation. Galpha i1K210E was insensitive to RGS16 but interacted with RGS16E89K. In rat uterine smooth muscle cells, wild type RGS16 abolished Gi-mediated alpha 2-adrenoreceptor signaling, whereas RGS16E89K was without effect. Both Galpha i1 and Galpha i1K210E mimicked the effect of alpha 2-adrenoreceptor stimulation. Galpha i1K210E was sensitive to RGS16E89K and 10-fold more potent than Galpha i1. Analogous mutants of Galpha q (Galpha qK215E) and RGS4 (RGS4E87K) were created and studied in COS-7 cells. The activity of wild type Galpha q was counteracted by wild type RGS4 but not by RGS4E87K. The activity of Galpha qK215E was inhibited by RGS4E87K, whereas non-mutated RGS4 was ineffective. We conclude that mutation of a conserved lysine residue to glutamate in Galpha i and Galpha q family members renders these proteins insensitive to wild type RGS proteins. Nevertheless, they are sensitive to glutamate to lysine mutants of RGS proteins. Such mutant pairs will be helpful tools in analyzing Galpha -RGS specificities in living cells

Coordinated control of sensitivity by two splice variants of Gαo in retinal ON bipolar cells

The high sensitivity of scotopic vision depends on the efficient retinal processing of single photon responses generated by individual rod photoreceptors. At the first synapse in the mammalian retina, rod outputs are pooled by a rod “ON” bipolar cell, which uses a G-protein signaling cascade to enhance the fidelity of the single photon response under conditions where few rods absorb light. Here we show in mouse rod bipolar cells that both splice variants of the Go α subunit, Gαo1 and Gαo2, mediate light responses under the control of mGluR6 receptors, and their coordinated action is critical for maximizing sensitivity. We found that the light response of rod bipolar cells was primarily mediated by Gαo1, but the loss of Gαo2 caused a reduction in the light sensitivity. This reduced sensitivity was not attributable to the reduction in the total number of Go α subunits, or the altered balance of expression levels between the two splice variants. These results indicate that Gαo1 and Gαo2 both mediate a depolarizing light response in rod bipolar cells without occluding each other’s actions, suggesting they might act independently on a common effector. Thus, Gαo2 plays a role in improving the sensitivity of rod bipolar cells through its action with Gαo1. The coordinated action of two splice variants of a single Gα may represent a novel mechanism for the fine control of G-protein activity

Locus coeruleus neuron growth cones and spinal cord regeneration

One of the major impediments to successful recovery of function after a spinal cord injury is thought to be the reaction of the neuronal growth cone to inhibitory influences in the local environment in or around the site of the injury. The growth cones of locus coeruleus neurons studied in vitro collapsed upon contact with an extract of CNS myelin but did not collapse on contact with an extract of PNS myelin. Coincident with the collapse of the growth cone, was an increase in internal free calcium concentration that was predominantly the result of an influx of calcium through channels in the plasma membrane. Omega-conotoxin, which specifically blocks N-type voltage-gated calcium channels, blocked both the myelin-induced calcium influx and the coincident collapse of the growth cone.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31931/1/0000884.pd

Hsp40 Couples with the CSPα Chaperone Complex upon Induction of the Heat Shock Response

In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPα). CSPα is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich “string” region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPα chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Gαs. In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPα complex. Association of Hsp40 with CSPα decreases CSPα-CSPα dimerization and enhances the CSPα-induced increase in steady state GTP hydrolysis of Gαs. This newly identified CSPα-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPα in neuroprotection

GoLoco motif proteins binding to Gαi1: insights from molecular simulations

Molecular dynamics simulations, computational alanine scanning and sequence analysis were used to investigate the structural properties of the Gαi1/GoLoco peptide complex. Using these methodologies, binding of the GoLoco motif peptide to the Gαi1 subunit was found to restrict the relative movement of the helical and catalytic domains in the Gαi1 subunit, which is in agreement with a proposed mechanism of GDP dissociation inhibition by GoLoco motif proteins. In addition, the results provide further insights into the role of the “Switch IV” region located within the helical domain of Gα, the conformation of which might be important for interactions with various Gα partners

Constitutive signaling of the Human Cytomegalovirus-encoded Chemokine receptor US28

Previously it was shown that the HHV-8-encoded chemokine receptor ORF74 shows considerable agonist-independent, constitutive activity giving rise to oncogenic transformation (Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C., and Cesarman, E. (1997) Nature 385, 347-350). In this study we report that a second viral-encoded chemokine receptor, the human cytomegalovirus-encoded US28, also efficiently signals in an agonist-independent manner. Transient expression of US28 in COS-7 cells leads to the constitutive activation of phospholipase C and NF-κB signaling via

G-protein signaling: back to the future

Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Gα·GDP/Gβγ heterotrimers to promote GDP release and GTP binding, resulting in liberation of Gα from Gβγ. Gα·GTP and Gβγ target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Gα and heterotrimer reformation — a cycle accelerated by ‘regulators of G-protein signaling’ (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this standard model. Whereas phospholipase C (PLC) β is activated by Gαq and Gβγ, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Gα nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways