N. Gautam

DIVERSITY OF G PROTEINS IN SIGNAL TRANSDUCTION

The heterotrimeric guanine nucleotide-binding proteins (G proteins) act as switches that regulate information processing circuits connecting cell surface receptors to a variety of effectors. The G proteins are present in all eukaryotic cells, and they control metabolic, humoral, neural, and developmental functions. More than a hundred different kinds of receptors and many different effectors have been described. The G proteins that coordinate receptor-effector activity are derived from a large gene family. At present, the family is known to contain at least sixteen different genes that encode the alpha subunit of the heterotrimer, four that encode beta subunits, and multiple genes encoding gamma subunits. Specific transient interactions between these components generate the pathways that modulate cellular responses to complex chemical signals.

The G-Protein βγ Complex

Abstract The vast majority of signalling pathways in mammalian cells are mediated by heterotrimeric (αβγ) G proteins. Reviewed here is regulation of signal transduction by the βγ complex at different protein interfaces: subunit–subunit, receptor–G protein and G protein–effector. The role of diverse β and γ subunit types in achieving specificity in signalling and potentially unidentified functions for these subunits also are discussed.

Efficient interaction with a receptor requires a specific type of prenyl group on the G protein gamma subunit.

Post-translational prenylation of the carboxyl-terminal cysteine is a characteristic feature of the guanine nucleotide-binding protein (G protein) γ subunits. Recent findings show that the farnesylated COOH-terminal tail of the γ1 subunit is a specific determinant of rhodopsin-transducin coupling. We show here that when synthetic peptides specific to the COOH-terminal tail of γ1 are chemically modified with geranyl, farnesyl, or geranylgeranyl groups and tested for their ability to interact with light activated rhodopsin, the farnesylated peptide is significantly more effective. These results show that an appropriate isoprenoid on the G protein γ subunit serves not only a membrane anchoring function but in combination with the COOH-terminal domain specifies receptor-G protein coupling.

G protein βγ complex-mediated apoptosis by familial Alzheimer's disease mutant of APP

In familial Alzheimers disease (FAD), three missense mutations, V642I, V642F and V642G, that co‐segregate with the disease phenotype have been discovered in the 695 amino acid form of the amyloid precursor protein APP. Expression of these mutants causes a COS cell NK1 clone to undergo pertussis toxin‐sensitive apoptosis in an FAD trait‐linked manner by activating the G protein Go, which consists of Gαo and Gβγ subunits. We investigated which subunit was responsible for the induction of apoptosis by V642I APP in NK1 cells. In the same system, expression of mutationally activated Gαo or Gαi induced little apoptosis. Apoptosis by V642I APP was antagonized by the overexpression of the carboxy‐terminal amino acids 495–689 of the β‐adrenergic receptor kinase‐1, which blocks the specific functions of Gβγ. Co‐transfection of Gβ2γ2 cDNAs, but not that of other Gβxγz (x = 1–3; z = 2, 3), induced DNA fragmentation in a manner sensitive to bcl‐2. These data implicate Gβγ as a cell death mediator for the FAD‐associated mutant of APP.

Shuttling of G Protein Subunits between the Plasma Membrane and Intracellular Membranes

Heterotrimeric G proteins (αβγ) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t½ < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

A DOMAIN ON THE G PROTEIN BETA SUBUNIT INTERACTS WITH BOTH ADENYLYL CYCLASE 2 AND THE MUSCARINIC ATRIAL POTASSIUM CHANNEL

The G protein βγ complex modulates the function of a variety of effectors in biological signaling. However, the individual roles of the β and γ subunits in this interaction are unknown. Unlike in the case of the α subunit, domains on the βγ complex that contact effectors have not yet been identified. We show here using the yeast two-hybrid system that the β subunit and not the γ subunit interacts with domains specific to adenylyl cyclase type 2 (AC2) and the muscarinic receptor-gated atrial inwardly rectifying potassium channel, GIRK1. Different β subunit types interact with these effector domains with different efficacies. Furthermore, an N-terminal fragment of 100 residues interacts with both these effector domains as effectively as the whole β subunit. This domain includes the region where the β subunit contacts with the α subunit in the crystal structure and may therefore explain the ability of the α subunit to shut off the activity of the βγ complex.

A family of G protein βγ subunits translocate reversibly from the plasma membrane to endomembranes on receptor activation.

The present model of G protein activation by G protein-coupled receptors exclusively localizes their activation and function to the plasma membrane (PM). Observation of the spatiotemporal response of G protein subunits in a living cell to receptor activation showed that 6 of the 12 members of the G protein γ subunit family translocate specifically from the PM to endomembranes. The γ subunits translocate as βγ complexes, whereas the α subunit is retained on the PM. Depending on the γ subunit, translocation occurs predominantly to the Golgi complex or the endoplasmic reticulum. The rate of translocation also varies with the γ subunit type. Different γ subunits, thus, confer distinct spatiotemporal properties to translocation. A striking relationship exists between the amino acid sequences of various γ subunits and their translocation properties. γ subunits with similar translocation properties are more closely related to each other. Consistent with this relationship, introducing residues conserved in translocating subunits into a non-translocating subunit results in a gain of function. Inhibitors of vesicle-mediated trafficking and palmitoylation suggest that translocation is diffusion-mediated and controlled by acylation similar to the shuttling of G protein subunits (Chisari, M., Saini, D. K., Kalyanaraman, V., and Gautam, N. (2007) J. Biol. Chem. 282, 24092–24098). These results suggest that the continual testing of cytosolic surfaces of cell membranes by G protein subunits facilitates an activated cell surface receptor to direct potentially active G protein βγ subunits to intracellular membranes.

KSR-1 Binds to G-protein βγ Subunits and Inhibits βγ-induced Mitogen-activated Protein Kinase Activation

The protein kinase KSR-1 is a recently identified participant in the Ras signaling pathway. The subcellular localization of KSR-1 is variable. In serum-deprived cultured cells, KSR-1 is primarily found in the cytoplasm; in serum-stimulated cells, a significant portion of KSR-1 is found at the plasma membrane. To identify the mechanism that mediates KSR-1 translocation, we performed a yeast two-hybrid screen. Three clones that interacted with KSR-1 were found to encode the full-length γ10 subunit of heterotrimeric G-proteins. KSR-1 also interacted with γ2and γ3 in a two-hybrid assay. Deletion analysis demonstrated that the isolated CA3 domain of KSR-1, which contains a cysteine-rich zinc finger-like domain, interacted with γ subunits. Coimmunoprecipitation experiments demonstrated that KSR-1 bound to β1γ3 subunits when all three were transfected into cultured cells. Lysophosphatidic acid treatment of cells induced KSR-1 translocation to the plasma membrane from the cytoplasm that was blocked by administration of pertussis toxin but not by dominant-negative Ras. Finally, transfection of wild-type KSR-1 inhibited β1γ3-induced mitogen-activated protein kinase activation in cultured cells. These results demonstrate that KSR-1 translocation to the plasma membrane is mediated, at least in part, by an interaction with βγ and that this interaction may modulate mitogen-activated protein kinase signaling.

Receptor-mediated Reversible Translocation of the G Protein βγ Complex from the Plasma Membrane to the Golgi Complex

Heterotrimeric G proteins have been thought to function on the plasma membrane after activation by transmembrane receptors. Here we show that, after activation by receptors, the G protein βγ complex selectively translocates to the Golgi. Receptor inactivation results in Gβγ translocating back to the plasma membrane. Both translocation processes occur rapidly within seconds. The efficiency of translocation is influenced by the type of γ subunit present in the G protein. Distinctly different receptor types are capable of inducing the translocation. Receptor-mediated translocation of Gβγ can spatially segregate G protein signaling activity.

STRUCTURAL DETERMINANTS FOR INTERACTION WITH THREE DIFFERENT EFFECTORS ON THE G PROTEIN BETA SUBUNIT

In the yeast two-hybrid system, a 100-residue fragment (β1A) from the N terminus of the β1 subunit interacts with domains specific to adenylyl cyclase 2 (AC2), the muscarinic atrial potassium channel (GIRK1), and phospholipase C-β2 (PLC-β2). Based on the crystal structure of the G protein, β1A is composed of an N-terminal α helix, a loop, and five β strands in which the C-terminal four β strands form a β sheet, the first of seven sheets that make up the propeller structure of the β subunit. A mutant of β1A (L4P, L7P, and L14P), in which the α helix was potentially destroyed, interacted poorly with the G protein γ subunit but effectively with domains of AC2, GIRK1, and PLC-β2. In contrast, another mutant of β1A (S72A, D76A, and W82A), in which a network of hydrogen bonds was disrupted, interacted poorly with GIRK1 and PLC-β2 domains, but effectively with the γ subunit and the AC2 domain. These results suggest that the proper folding of the first five β strands in the G protein β subunit is a requirement for appropriately positioning residues that interact with GIRK1 and PLC-β2. Furthermore, since mutations that potentially disrupted the folding of these β strands did not affect interaction with AC2, the structural determinants on the G protein β subunit for interaction with various effectors may be different.