AMP-activated protein kinase (AMPK) acts as an energy sensor, being activated by metabolic stresses and regulating cellular metabolism. AMPK is a heterotrimer consisting of a catalytic ␣ subunit and two regulatory subunits, ␤ and ␥. It had been reported that the mammalian AMPK ␣ subunit contained an autoinhibitory domain (␣1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the ␣ subunit (␣1-(313-335)), which includes a predicted ␣-helix, is responsible for ␣ subunit autoinhibition. The role of the residues in this segment for autoinhibition was further investigated by systematic site-directed mutation. Several hydrophobic and charged residues, in particular Leu-328, were found to be critical for ␣1 autoinhibition. An autoinhibitory structural model of human AMPK ␣1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 Å and may stabilize the autoinhibitory conformation. Further mutation analysis showed that V298G mutation significantly activated the kinase activity. Moreover, the phosphorylation level of acetyl-CoA carboxylase, the AMPK downstream substrate, was significantly increased in COS7 cells overexpressing AMPK ␣1-(1-394) with deletion of residues 313-335 (⌬␣394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with ⌬␣394, V298G, or L328Q mutants, which indicated that these AMPK ␣1 mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK ␣ autoinhibitory function.The AMP-activated protein kinase (AMPK) 2 is a sensor of cellular energy state, being activated by a large variety of cellular stresses that increase cellular AMP and decrease ATP levels, such as glucose deprivation, hypoxia, oxidative stress, heat shock, and ischemia (1-3). AMPK is also activated by physiological stimuli, such as exercise, muscle contraction, hormones like leptin and adiponectin, pharmacological agents like thiazolidinediones and metformin, and a widely used AMPK activator, 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (4 -10), modulating multiple metabolic pathways (11). Therefore, AMPK has been investigated for the treatment of type II diabetes, obesity, and even cancer (12).AMPK is a heterotrimeric serine/threonine protein kinase consisting of a catalytic ␣ subunit and two regulatory subunits, ␤ and ␥ (13). In mammals, each AMPK subunit has multiple isoforms, ␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3 (14), suggesting that multiple heterotrimeric complexes may exist in different tissues and play different roles. Optimal kinase activity requires the formation of a heterotrimeric complex, involving AMP allosteric activation, and phosphorylation on Thr 172 within the activation loop of the catalytic ␣ subunit by an upstream kinase, AMPK kinase (AMPKK), identified as LKB1 or calcium/calmodulin-dependent protein kinase kinase ␤ (CAMKK␤) (13,(15)(16)(17)(18)(19)(20...

The type 2 bradykinin receptor (B2R) is a G protein-coupled receptor (GPCR) in the cardiovascular system, and the dysfunction of B2R leads to inflammation, hereditary angioedema, and pain. Bradykinin and kallidin are both endogenous peptide agonists of B2R, acting as vasodilators to protect the cardiovascular system. Here we determine two cryo-electron microscopy (cryo-EM) structures of human B2R-Gq in complex with bradykinin and kallidin at 3.0 Å and 2.9 Å resolution, respectively. The ligand-binding pocket accommodates S-shaped peptides, with aspartic acids and glutamates as an anion trap. The phenylalanines at the tail of the peptides induce significant conformational changes in the toggle switch W2836.48, the conserved PIF, DRY, and NPxxY motifs, for the B2R activation. This further induces the extensive interactions of the intracellular loops ICL2/3 and helix 8 with Gq proteins. Our structures elucidate the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins coupling of B2R.

Type 2 bradykinin receptor (B2R) is an essential G protein-coupled receptor (GPCR) that regulates the cardiovascular system as a vasodepressor. Dysfunction of B2R is also closely related to cancers and hereditary angioedema (HAE). Although several B2R agonists and antagonists have been developed, icatibant is the only B2R antagonist clinically used for treating HAE. The recently determined structures of B2R have provided molecular insights into the functions and regulation of B2R, which shed light on structure-based drug design for the treatment of B2R-related diseases. In this review, we summarize the structure and function of B2R in relation to drug discovery and discuss future research directions to elucidate the remaining unknown functions of B2R dimerization.