The beta3-adrenergic receptor (beta3AR) is a G protein-coupled receptor (GPCR) expressed predominantly in adipose tissue, where it mediates the effects of noradrenaline-stimulated lipolysis in white adipose tissue (WAT) and thermogenesis in brown adipose tissue (BAT). Selective agonists for the beta3AR can prevent or reverse obesity and insulin resistance in canine and rodent models, and are under pharmaceutical development for humans. In addition to potent stimulation of lipolysis, beta3AR agonists promote the appearance of thermogenically active brown adipocytes in typical white adipose depots but the mechanism responsible for this effect is not understood. Our preliminary data show that the beta3AR in adipocytes is constitutively coupled to both Galphas and Galphai, leading to the activation of the protein kinase A (PKA) and MAP kinase (ERK1/2) pathways, respectively ERK1/2 activation is sensitive to pertussis toxin, dependent upon an activated receptor tyrosine kinase (RTK), and leads to the formation of a signaling complex containing beta3AR, an RTK, and c-src. The beta3AR is not subject to phosphorylation-induced desensitization, beta-arrestin binding, internalization and recycling: features required by other GPCRs to trigger ERK1/2 activation. Instead, additional preliminary data suggest that peptide motifs in the receptor confer a novel mechanism of ERK activation by a GPCR. Two main hypotheses are proposed. First, that unique structural features of the beta3AR containing proline rich (PXXP) motifs within intracellular domains of the receptor serve directly as docking sites for the assembly of RTK and src- dependent signaling complexes. Second, that the confluence of PKA and ERK activation controls the efficacy of lipid mobilization and proliferation/differentiation of brown adipocytes and thermogenic respiration.
Three aims are proposed to test these hypotheses. (1) Establish identity and activity of intracellular proteins required for ERK activation by beta3AR. (2) Determine whether the proline rich (PXXP) motifs within the third cytoplasmic loop and carboxyl terminus of the receptor function as a scaffold for recruitment and activation of src or other SH3-domain containing proteins, and whether these regions are necessary and sufficient to elicit ERK1/2 activation. (3) Cell lines and transgenic animals expressing wild-type beta3AR or mutated forms of beta3AR that lack the ability to activate ERK1/2 will be generated. The effect of selective beta3AR agonists to stimulate lipolysis in white adipocytes, UCP1 transcription and thermogenesis in brown adipocytes, and the physiological effects of beta3AR-agonists in vivo on glycemic control, obesity, and the appearance of brown adipocytes in white fat depots will be determined.
