Insight into the Brain-Specific Alpha Isoform of the Scaffold Protein SH2B1 and its Rare Obesity-Associated Variants

Abstract

Obesity poses a major health problem since it increases the risk for type 2 diabetes, metabolic syndrome, heart disease, and cancer. Mutations in SH2B1 have been identified in patients exhibiting severe childhood obesity and insulin resistance. Mice deficient in SH2B1 exhibit a similar phenotype to the patients, suggesting an important role for SH2B1 in regulating energy homeostasis. SH2B1 is alternatively spliced, leading to four isoforms (alpha, beta, gamma, and delta) that share an N-terminal 631 amino acids but have unique C-terminal tails. The findings that all SH2B1 isoforms are expressed in the brain and neuronal expression of SH2B1 beta rescues the obese phenotype in SH2B1-deficient mice suggest that SH2B1 plays an important function in neurons. In further support of a neuronal function for SH2B1, SH2B1 enhances neurotrophic factor-induced neurite outgrowth and the human obesity-associated mutations impair the ability of SH2B1 to promote neurite outgrowth. To gain insight into critical functions of SH2B1, its isoforms, and the human obesity-associated mutations, the work in this thesis characterizes the ability of rare SH2B1 mutations associated with human obesity both shared by all isoforms and those specific to SH2B1 alpha to affect cellular actions of SH2B1. It further examines how the unique C-terminal tails of SH2B1 regulate the actions of the shared 631 N-terminal amino acids of SH2B1.

Our collaborator I. S. Farooqi identified variants in SH2B1 encoding for R227C, G238C, R270W, E299G, or T546A and the SH2B1 alpha-specific mutations A663V, V695M, or A723V in individuals with severe-early onset obesity and insulin resistance. We determined that like SH2B1 beta, SH2B1 alpha enhanced growth hormone-stimulated motility of macrophages and the mutations T546A, A663V and A723V impaired that enhancement. Unlike SH2B1 beta, SH2B1 alpha was unable to enhance NGF-mediated neurite outgrowth and the mutations in SH2B1 alpha had no impact. However, mutations G238C, R270W, E299G and T546A impaired SH2B1 beta-enhancement of NGF-induced neurite outgrowth. This led us to conclude that variants can disrupt specific isoform function, suggesting novel regulatory roles in SH2B1 isoform function. The discrepancy between SH2B1 alpha and beta regulation of neurite outgrowth directed us to ask how the unique C-terminal tails of the alpha and beta isoforms affect the ability of SH2B1 to regulate NGF-induced neurite outgrowth. By comparing the actions of SH2B1 alpha and beta to those of the N-terminal 631 amino acids shared by all isoforms of SH2B1, we found that the alpha tail prevents: 1) the ability of SH2B1 to cycle through the nucleus, and 2) SH2B1 enhancement of NGF-induced neurite outgrowth, gene expression, and phosphorylation of NGF-receptor, TrkA, and downstream signaling proteins Akt and PLC gamma. These functions were restored when Tyr753 in the alpha tail was mutated to Phe, suggesting that phosphorylation of Tyr753 regulates SH2B1 alpha function. We provide evidence that TrkA phosphorylates Tyr55 and Tyr439 in both SH2B1 alpha and SH2B1 beta, and Tyr753 in SH2B1 alpha. Finally, co-expression of SH2B1 alpha inhibits the ability of SH2B1 beta to enhance NGF-induced neurite outgrowth. These results suggest that the C-terminal tails of SH2B1 isoforms are key determinants of their cellular roles and are regulated by phosphorylation. By providing valuable information about how seemingly minor differences between isoforms can have a profound impact on the function of that protein, my studies also provide important insight into the impact of differential splicing on neuron function.