Abstract

Overexpression of Gα13, a member of the G12/13 subfamily of heterotrimeric G proteins, has been implicated in cell transformation and the progression of several cancer types. The mechanism through which wildtype, overexpressed Gα13 drives aberrant growth signaling is not known. G protein α subunits are modified post-translationally by acylation at the N-terminus, and this addition of a palmitoyl and/or myristoyl group occurs for all mammalian Gα proteins. Gα13 was previously engineered to mutate two cysteine residues necessary for dual palmitoylation. This modification abrogated signaling to serum response factor by Gα13 in both its wildtype and constitutively activated forms. To determine whether myristoylation restores signaling to non-acylated wildtype Gα13, the protein was modified to harbor the 6 N-terminal amino acids from GαT, which is myristoylated but not palmitoylated. Loss of growth signaling by non-palmitoylated, wildtype Gα13 was rescued by introduction of this myristoylated sequence. In addition, we used cellular fractionation to assess intracellular distribution of these Gα13 variants. Overexpressed, wildtype Gα13 showed a shift from a membrane associated fraction to a soluble pool, which correlated with a sharp increase in serum response factor signaling. Gα13 lacking any acylation sites localized entirely to the soluble fraction but exhibited no growth signaling. To query the nucleotide bound state of the soluble Gα13 pool, we combined cell fractionation with trypsin digestion assays. Surprisingly, overexpressed wildtype Gα13 in the soluble fraction was fully degraded, suggesting lack of GTP binding. These constructs have facilitated co-precipitation experiments to determine whether change in acylation state affects Gα13 binding to specific target proteins. E-cadherin and the RhoGEFs p115, PDZ and LARG were tested for their ability to bind Gα13 variants. Non-palmitoylated, constitutively active (QL) Gα13 was pulled down by all four proteins, evidence that its signal abrogation may be due to different cellular localization rather than reduced binding affinity. The wildtype version of this Gα13 construct appears to lose affinity to E-cadherin, though these results need to be explored further. Also examined was the role of an N-terminal polybasic motif in overexpressed wildtype Gα13 signaling, through amino acid substitutions in this region. Mutants were constructed using a non-palmitoylated variant of Gα13, that inserted nine lysines upstream of the polybasic region. These constructs, called 9K, contained only an exaggerated positive region as a membrane signal and yet the QL type signaled almost as strongly as the positive control. Lastly, 3Q mutants were constructed to reduce the number of positively charged amino acids in the PBM region by converting them to glutamine. The wildtype version of this construct lost 80% of signal strength while QL was unaffected. These experiments demonstrate that rather than lipid membranes being a passive environment on which the dance of GPCR signaling occurs, lipid-protein interactions for Gα13 are crucial for its capacity to ability to signal through SRE.

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