Regulation, Signaling, and Physiological Functions of G-Proteins.

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.

Members of the heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) family play key roles in many physiological functions and are extensively exploited pharmacologically to treat diseases. Many of the diverse effects of individual GPCRs on cellular physiology are transduced by heterotrimeric G proteins, which are composed of α, β, and γ subunits. GPCRs interact with and stimulate the binding of guanosine triphosphate (GTP) to the α subunit to initiate signaling. Mammalian genomes encode 16 different G protein α subunits, each one of which has distinct properties. We developed a single-platform, optical strategy to monitor G protein activation in live cells. With this system, we profiled the coupling ability of individual GPCRs for different α subunits, simultaneously quantifying the magnitude of the signal and the rates at which the receptors activated the G proteins. We found that individual receptors engaged multiple G proteins with varying efficacy and kinetics, generating fingerprint-like profiles. Different classes of GPCR ligands, including full and partial agonists, allosteric modulators, and antagonists, distinctly affected these fingerprints to functionally bias GPCR signaling. Finally, we showed that intracellular signaling modulators further altered the G protein-coupling profiles of GPCRs, which suggests that their differential abundance may alter signaling outcomes in a cell-specific manner. These observations suggest that the diversity of the effects of GPCRs on cellular physiology may be determined by their differential engagement of multiple G proteins, coupling to which produces signals with varying signal magnitudes and activation kinetics, properties that may be exploited pharmacologically.

/pdf/distinct-profiles-of-functional-discrimination-among-g-42fyxe13u9.pdf

Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors

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

Ric-8 proteins are molecular chaperones that direct nascent G protein α subunit membrane association.

Ric-8B Is a GTP-dependent G Protein αs Guanine Nucleotide Exchange Factor

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

Method and apparatus for analyzing the deformation of an object resulting from the application of stress. A novel optical element is used to perform shearometric analysis upon a test object. The novel optical element has an overall pattern of first and second pluralities of regions having significantly different indices of transmissivity. The pattern of variations causes pairs of light rays which are reflected from two distinct points on the test object at a divergent angle to emerge from the optical element so that they are nearly parallel. The nearly parallel rays are then received upon a photoelectrical sensing means such as a video camera or photoelectric array. Because the rays impinging thereon are nearly parallel, they may be adequately resolved by the photoelectric sensing means, thus enabling shearometric analysis to be performed electronically in a rapid and cost-effective manner.

Shearing optical element for interferometric analysis system

A shearogram representing an object and including a fringe pattern arising from a change in local surface strain brought about by an applied stress during the making of the shearogram is reconstructed by a special viewer. The viewer holds the shearogram transparency adjacent to a focusing lens having an optical axis, and projects off-axis white light through the lens and transparency to a focal plane where the resulting diffraction pattern is viewed directly on a screen or by a video camera and display monitor. By projecting the light from different positions around the axis a first position can be found which tunes the diffraction efficiency to an optimum for the best view of fringe patterns and a second position can be found which eliminates the fringe patterns and shows only a photographic image of the object. The light source is a rotatable disk with two apertures containing light diffusing elements which are illuminated by a lamp. The disk is rotated to change the projection angle.

Forrest A. Wright

Papers

Ric-8 proteins are molecular chaperones that direct nascent G protein α subunit membrane association.

Ric-8B Is a GTP-dependent G Protein αs Guanine Nucleotide Exchange Factor

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

Shearing optical element for interferometric analysis system

Method and apparatus for viewing a shearographic image