Eva J. Neer

Heterotrimeric C proteins: Organizers of transmembrane signals

Hundreds of chemical and physical signals constantly bombard the surface of all cells. Some of these do not enter the cell but, instead, bind to receptors at the cell surface and initiate a flow of information that moves to the cell interior. The receptors for many hormones (such as catecholamines, gonadotropins, parathyroid hormone, and glucagon), odorants, and light span the membrane seven times (reviewed by Dohlman et al., 1991). Stimulation of these receptors activates a group of coupling proteins (called G proteins because they bind GTP) that regulate a variety of enzymes and ion channels. The target enzymes or ion channels are called effectors because changes in their activity cause the changes in ionic composition or in second messenger levels (such as cAMP or inositol phosphate levels) that ultimately lead to the cellular response. Every eukaryotic cell contains receptors for many kinds of chemical and/or physical signals, many different types of G proteins, and many effectors, each with multiple subtypes. A cell can only respond to those signals for which it has a receptor, but the specificitywith which the receptor interacts with the coupling proteins (the G proteins) defines the range of responses that a cell is able to make. Receptors are highly selective for their ligands. If a receptor can interact with only one subtype of G protein that can, in turn, activate only one type of effector, the response will be very focused. In contrast, if a receptor can interact with several G proteins, each of which can interact with more than one effector, the response would be expected to be spread over several pathways. As will be d iscussed below, a cell may respond to some signals with a very defined set of actions, but may respond to others less specifically. Similarly, a ligand that gives a focused response in one cell may cause a pleiotypic response in another. Over the last decade, there has been enormous progress in defining the elements that are involved in transmembrane signaling. A very large number of receptors have been cloned, characterized, and subdivided into families. Four subfamilies of G protein (~ subunits have been defined, and multiple G protein 13 and y subunits have been identified. We now know that effectors often come in several subtypes, each with different regulatory properties. What is still mysterious is exactly what determines specificity of the response of a cell to an extracellular stimulus. What is the grammar that controls the interpretation of signals? In this review, I will summarize some features of the structure and function of mammalian G protein subunits, then discuss how the elements of the cellular language may be ordered and weighted to allow the cell to respond properly to the message.

The WD repeat: a common architecture for diverse functions.

Our knowledge of the large family of proteins that contain the WD repeat continues to accumulate. The WD-repeat proteins are found in all eukaryotes and are implicated in a wide variety of crucial functions. The solution of the three-dimensional structure of one WD-repeat protein and the assumption that the structure will be common to all members of this family has allowed subfamilies of WD-repeat proteins to be defined on the basis of probable surface similarity. Proteins that have very similar surfaces are likely to have common binding partners and similar functions.

G protein beta gamma subunits

Guanine nucleotide binding (G) proteins relay extracellular signals encoded in light, small molecules, peptides, and proteins to activate or inhibit intracellular enzymes and ion channels. The larger G proteins, made up of G heterotrimers, dissociate into G and G subunits that separately activate intracellular effector molecules. Only recently has the G subunit been recognized as a signal transduction molecule in its own right; G is now known to directly regulate as many different protein targets as the G subunit. Recent X-ray crystallography of G ,G , and G subunits will guide the investigation of structure-function relationships.

G Protein Heterodimers: New Structures Propel New Questions

The docking of G␣ to G␤␥ involves extensive contacts: *Department of Medicine binding of the G␣ N-terminal ␣ helix to the side of the Cardiovascular Division G␤ propeller parallel to its central tunnel and binding of Brigham and Womens Hospital the catalytic domain of G␣ to the top surface of the ␤ and Harvard Medical School propeller (see Figure 1). Removal of the N-terminal ␣ Boston, Massachusetts 02115 helix from G␣ prevents formation of G␣␤␥ heterotrimers † Department of Pharmacology (reviewed by Neer, 1995). The catalytic domain binds to and Biomolecular Engineering Research Center G␤ through a region called switch II, previously known Boston University to change conformation upon nucleotide binding and to Boston, Massachusetts 02215 be chemically cross-linkable to the G␤␥ subunit (Garcia-Higuera et al., 1996, and references therein). A number of salt bridges and the fit of G␤ Trp-99 into a hydrophobic The heterotrimeric G proteins transmit signals from a pocket on G␣ determine binding of G␣ to the top of the variety of cell surface receptors to enzymes and chan-propeller. Mutation of an equivalent Trp in yeast G␤ nels (reviewed by Neer, 1995). The heterotrimer consists disrupts G␤␥ binding to G␣, leading to constitutive acti-of an ␣ subunit that binds and hydrolyzes GTP and a vation of the yeast mating pathway (see Wall et al., 1995; pair of proteins, ␤ and ␥, that are tightly associated with Lambright et al., 1996). each other. The GTP-activated G␣ subunits dissociate GDP-liganded G␣ in a G␣␤␥ heterotrimer is different from G␤␥, and both subunits then activate their respec-from GDP-liganded G␣ alone (see references in Wall et tive effectors. The subunits stay separated until GTP is al., 1995; Lambright et al., 1996). This may be partly due hydrolyzed to GDP, whereupon they reassemble and to differences in crystal structures and crystal–crystal both become inactive. Therefore, the contact surface between G␣ and G␤␥ has major regulatory importance. Two groups have now independently determined the The new structures (Figure 1) reveal that the conformation of the GDP-liganded ␣ subunit in the heterotrimer is different from the GDP-liganded ␣ subunit alone (and also different from the crystallized GTP␥S-liganded form). They also reveal that the G␤ subunit folds into a highly symmetric ␤ propeller. Each propeller blade consists of a small four-stranded twisted ␤ sheet, the innermost ␤ strand being nearly parallel to the axis of the central tunnel (see schematic in Figure 2). This …

Cardiac myocytes express mRNA for ten RGS proteins: changes in RGS mRNA expression in ventricular myocytes and cultured atria

Regulators of G‐protein signalling (RGS) are recently identified proteins that shorten the lifetime of the activated G protein. We now show that rat cardiac myocytes express mRNA for at least 10 RGS. The mRNA for RGS‐r is barely detectable in rat ventricles, but increases more than 20‐fold during the 60‐ to 90‐min process of isolating ventricular myocytes, and after 90 min of culture of atrial pieces in medium with Ca2+. Both in myocytes and in atria, the rise in RGS‐r is transient. The mRNA for cardiac RGS5, but not RGS‐r, is developmentally regulated. These studies suggest that rapid regulation of RGS levels may be a new mechanism that governs how signals are transmitted across the cardiac cell membrane.

FOLDING A WD REPEAT PROPELLER : ROLE OF HIGHLY CONSERVED ASPARTIC ACID RESIDUES IN THE G PROTEIN BETA SUBUNIT AND SEC13

The β subunit of the heterotrimeric G proteins that transduce signals across the plasma membrane is made up of an amino-terminal α-helical segment followed by seven repeating units called WD (Trp-Asp) repeats that occur in about 140 different proteins. The seven WD repeats in Gβ, the only WD repeat protein whose crystal structure is known, form seven antiparallel β sheets making up the blades of a toroidal propeller structure (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell83, 1047–1058; Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 369–374). It is likely that all proteins with WD repeats form a propeller structure. Alignment of the sequence of 918 unique WD repeats reveals that 85% of the repeats have an aspartic acid (D) residue (not the D of WD) in the turn connecting β strands b and c of each putative propeller blade. We mutated each of these conserved Asp residues to Gly individually and in pairs in Gβ and in Sec13, a yeast WD repeat protein involved in vesicular traffic, and then analyzed the ability of the mutant proteins to fold in vitro and in COS-7 cells. In vitro, most single mutant Gβ subunits fold into Gβγ dimers more slowly than wild type to a degree that varies with the blade. In contrast, all single mutants form normal amounts of Gβγ in COS-7 cells, although some dimers show subtle local distortions of structure. Most double mutants assemble poorly in both systems. We conclude that the conserved Asp residues are not equivalent and not all are essential for the folding of the propeller structure. Some may affect the folding pathway or the affinity for chaperonins. Mutations of the conserved Asp in Sec13 affect folding equally in vitro and in COS-7 cells. The repeats that most affected folding were not at the same position in Sec13 and Gβ. Our finding, both in Gβ and in Sec13, that no mutation of the conserved Asp entirely prevents folding suggests that there is no obligatory folding order for each repeat and that the folding order is probably not the same for different WD repeat proteins, or even necessarily constant for the same protein.

THE G PROTEIN GAMMA SUBUNIT : REQUIREMENTS FOR DIMERIZATION WITH BETA SUBUNITS

Guanine nucleotide-binding protein β and γ subunits form a tightly bound complex that can only be separated by denaturation. Assembly of β and γ subunits is a complicated process. The β1 and γ2 subunits can be synthesized in vitro in rabbit reticulocyte lysate and then assembled into dimers, but β1 cannot form βγ dimers when synthesized in a wheat germ extract. In contrast, γ2 translated in either system can dimerize with β1, suggesting that dimerization-competent γ2 can be synthesized without the aid of specific chaperonins or other cofactors. Dimerization-competent γ2 in solution forms an asymmetric particle with a Stokes radius of about 21 ± 0.4 Å (n = 4), s20,w of 0.9 S (range 0.8-1.0 S, n = 2), and frictional ratio of 1.57 (assuming no hydration). To define the part of γ2 that is needed for native βγ dimer formation, a series of N- and C-terminal truncations were generated, synthesized in vitro, and incubated with β1. Dimerization was assessed by stabilization of β1 to tryptic proteolysis. Truncation of up to 13 amino acids at the C terminus did not affect dimerization with β1, whereas removal of 27 amino acids prevented it. Therefore, a region between residues 45 and 59 of γ2 is important for dimerization. Truncation of 15 amino acids from the N terminus greatly diminished the formation of βγ dimers, while removal of 25 amino acids entirely blocked it. Thus, another region important for forming native βγ is near the N terminus. Extension of the N terminus by 12 amino acids that include the influenza virus hemagglutinin epitope did not prevent βγ dimerization. Furthermore, in intact 35S-labeled COS cells, epitope-tagged γ2 coimmunoprecipitates with β and α subunits. The N-terminal epitope tag must lie at the surface of the heterotrimer since it prevents neither heterotrimer formation nor access of the antibody.

Signal Transduction in Atria and Ventricles of Mice With Transient Cardiac Expression of Activated G Protein αq

We recently showed that the transient expression of a hemagglutinin (HA) epitope-tagged, constitutively active mutant of the G protein alpha(q) subunit (HAalpha(q)*) in the hearts of transgenic mice is sufficient to induce cardiac hypertrophy and dilatation that continue to progress after HAalpha(q)* protein becomes undetectable. We demonstrated that the activity of phospholipase Cbeta, the immediate downstream target of activated Galpha(q), is increased at 2 weeks, when HAalpha(q)* is expressed, but also at 10 weeks, when HAalpha(q)* is no longer detectable. This observation suggested that the transient HAalpha(q)* expression causes multiple, persistent changes in cellular signaling pathways. We now demonstrate changes in the level, activity, or both of several signaling components, including changes in the amount and hormone responsiveness of phospholipase Cbeta enzymes, in the basal level of diacylglycerol (which predominantly reflects activation of phospholipase D), in the amount or distribution of protein kinase C (PKC) isoforms (PKCalpha, PKCdelta, and PKCepsilon), and in the amount of several endogenous G proteins. These changes vary depending on the isoform of the signaling molecule, the chamber in which it is expressed, and the presence or absence of HAalpha(q)*. Our results suggest that a network of linked signaling functions determines the development of hypertrophy. They also suggest that atria and ventricles represent different signaling domains. It is likely that such changes occur in other model systems in which the activity of a single signaling component is increased, either due to an activating mutation or due to overexpression of the wild type.

Signal transduction through G proteins in the cardiac myocyte.

The membrane of the cardiocyte contains a large variety of molecules whose function is to transmit signals from outside the cell to inside. The signals are initiated by receptors for a variety of agonists and propagated by a family of heterotrimeric G proteins to ion channels and intracellular enzymes. The large complement of receptors, G proteins, and effectors found in the cardiocyte raises fundamental questions about the mechanisms that assure the precision and timing of the heart cells response to external stimuli.

Intersubunit surfaces in G protein alpha beta gamma heterotrimers. Analysis by cross-linking and mutagenesis of beta gamma.

Heterotrimeric guanine nucleotide binding proteins (G proteins) are made up of α, β, and subunits, the last two forming a very tight complex. Stimulation of cell surface receptors promotes dissociation of α from the β dimer, which, in turn, allows both components to interact with intracellular enzymes or ion channels and modulate their activity. At present, little is known about the conformation of the β dimer or about the areas of β that interact with α. Direct information on the orientation of protein surfaces can be obtained from analysis of chemically cross-linked products. Previous work in this laboratory showed that 1,6-bismaleimidohexane, which reacts with cysteine residues, specifically cross-links α to β and β to (Yi, F., Denker, B. M., and Neer, E. J.(1991) J. Biol. Chem. 266, 3900-3906). To identify the residues in β and involved in cross-linking to each other or to α, we have mutated the cysteines in β, , and and analyzed the mutated proteins by in vitro translation in a rabbit reticulocyte lysate. All the mutants were able to form β dimers that could interact with the α subunit. We found that 1,6-bismaleimidohexane can cross-link β to but not to . The cross-link goes from Cys in β to Cys in . This cysteine is absent from any of the other known isoforms and therefore confers a distinctive property to . The β subunit in the β dimer can be cross-linked to an unidentified protein in the rabbit reticulocyte lysate, generating a product slightly larger than cross-linked β. The β subunit can also be cross-linked to α, giving rise to two products on SDS-polyacrylamide gel electrophoresis, both of which were previously shown to be formed by cross-linking β to Cys in α (Thomas, T. C., Schmidt, C. J., and Neer, E. J.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10295-10299). Mutation of Cys in β abolished one of these two products, whereas mutation of Cys abolished the other. Because both α-β cross-linked products are formed in approximately equal amounts, Cys and Cys in β are equally accessible from Cys in α. Our findings begin to define intersubunit surfaces, and they pose structural constraints upon any model of the β dimer.