The large class of adhesion G protein-coupled receptors (aGPCRs) bind extracellular matrix or neighboring cell-surface ligands to regulate organ and tissue development through an unknown activation mechanism. We examined aGPCR activation using two prototypical aGPCRs, GPR56 and GPR110. Active dissociation of the noncovalently bound GPR56 or GPR110 extracellular domains (ECDs) from the respective seven-transmembrane (7TM) domains relieved an inhibitory influence and permitted both receptors to activate defined G protein subtypes. After ECD displacement, the newly revealed short N-terminal stalk regions of the 7TM domains were found to be essential for G protein activation. Synthetic peptides comprising these stalks potently activated GPR56 or GPR110 in vitro or in cells, demonstrating that the stalks comprise a tethered agonist that was encrypted within the ECD. Establishment of an aGPCR activation mechanism provides a rational platform for the development of aGPCR synthetic modulators that could find clinical utility toward aGPCR-directed disease.

https://www.pnas.org/content/pnas/112/19/6194.full.pdf

Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.

Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/i. The mini-G proteins were shown to couple to relevant GPCRs and to form stable complexes with purified receptors that could be purified by size exclusion chromatography. Agonist-bound GPCRs coupled to a mini-G protein showed higher thermal stability compared to the agonist-bound receptor alone. Fusion of GFP at the N-terminus of mini-G proteins allowed receptor coupling to be monitored by fluorescence-detection size exclusion chromatography (FSEC) and, in a separate assay, the affinity of mini-G protein binding to detergent-solubilised receptors was determined. This work provides the foundation for the development of any mini-G protein and, ultimately, for the structure determination of GPCRs in a fully active state.

/pdf/mini-g-proteins-novel-tools-for-studying-gpcrs-in-their-2byjsqpsnc.pdf

Mini-G proteins: Novel tools for studying GPCRs in their active conformation.

Adenylyl cyclases (ACs) generate the second messenger cAMP from ATP. Mammalian cells express nine transmembrane AC (mAC) isoforms (AC1–9) and a soluble AC (sAC, also referred to as AC10). This review will largely focus on mACs. mACs are activated by the G-protein Gαs and regulated by multiple mechanisms. mACs are differentially expressed in tissues and regulate numerous and diverse cell functions. mACs localize in distinct membrane compartments and form signaling complexes. sAC is activated by bicarbonate with physiologic roles first described in testis. Crystal structures of the catalytic core of a hybrid mAC and sAC are available. These structures provide detailed insights into the catalytic mechanism and constitute the basis for the development of isoform-selective activators and inhibitors. Although potent competitive and noncompetitive mAC inhibitors are available, it is challenging to obtain compounds with high isoform selectivity due to the conservation of the catalytic core. Accordingly, caution must be exerted with the interpretation of intact-cell studies. The development of isoform-selective activators, the plant diterpene forskolin being the starting compound, has been equally challenging. There is no known endogenous ligand for the forskolin binding site. Recently, development of selective sAC inhibitors was reported. An emerging field is the association of AC gene polymorphisms with human diseases. For example, mutations in the AC5 gene (ADCY5) cause hyperkinetic extrapyramidal motor disorders. Overall, in contrast to the guanylyl cyclase field, our understanding of the (patho)physiology of AC isoforms and the development of clinically useful drugs targeting ACs is still in its infancy.

https://pharmrev.aspetjournals.org/content/pharmrev/69/2/93.full.pdf

International union of basic and clinical pharmacology. CI. structures and small molecule modulators of mammalian adenylyl cyclases

Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide–binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A – or Ric-8B –deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A −/− ES cells, which resulted from reduced steady-state amounts of Gα i , Gα q , and Gα 13 proteins to s and total Gβ protein were partially reduced in Ric-8A −/− cells compared to those in wild-type cells, and only the amount of Gα s was reduced substantially in Ric-8B −/− cells. The abundances of mRNAs encoding the G protein α subunits were largely unchanged by loss of Ric-8A or Ric-8B . The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous Gα i and Gα q were efficiently translated in Ric-8A −/− cells but integrated into endomembranes poorly; however, the reduced amounts of G protein α subunits that reached the membrane still bound to nascent Gβγ. Finally, Gα i , Gα q , and Gβ 1 proteins exhibited accelerated rates of degradation in Ric-8A −/− cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent Gα subunits with cellular membranes.

/pdf/ric-8-proteins-are-molecular-chaperones-that-direct-nascent-2a8jel6w10.pdf

Wei Kan

Papers

Purification of Heterotrimeric G Protein α Subunits by GST-Ric-8 Association: PRIMARY CHARACTERIZATION OF PURIFIED Gαolf*