Abstract
Microtubules (MTs), a major component of cell cytoskeleton, exhibit diverse cellular functions including cell motility, intracellular transport, cell division, and differentiation. These functions of MTs are critically dependent on their ability to polymerize and depolymerize. Although a significant progress has been made in identifying cellular factors that regulate microtubule assembly and dynamics, the role of signal transducing molecules in this process is not well understood. It has been demonstrated that heterotrimeric G proteins, which are components of G protein-coupled receptor (GPCR) signaling pathway, interact with microtubules and play important roles in regulating assembly/dynamics of this cytoskeletal filament. While α subunit of G proteins (Gα) inhibits microtubule assembly and accelerates microtubule dynamics, Gβγ promotes tubulin polymerization. In this chapter, we review the current status of G-protein modulation of microtubules and cellular and physiological aspects of this regulation. Molecular, biochemical, and cellular methodologies that have been used to advance this field of research are discussed. Emphasis has been given on G-protein-microtubule interaction in neuronal differentiation as significant progress has been made in this field. The outcome from this research reflects the importance of GPCRs in transducing extracellular signals to regulate a variety of microtubule-associated cellular events.
Keywords
- cytoskeleton
- G-proteins
- microtubules
- neuronal differentiation
- Gβγ
- tubulin
- G protein-coupled receptor
- GTP-binding proteins
1. Introduction
The major component of microtubules (MTs) is the heterodimeric protein tubulin, consisting of α and β subunits, which are assembled into linear protofilaments. The protofilaments associate laterally to form the microtubule, a 25-nm-wide hollow cylindrical polymeric structure [1]. Due to the asymmetry of the αβ-tubulin heterodimer, MTs are polar structures with two distinct ends. These ends possess different polymerization rates: a slow-growing minus end with an exposed α-tubulin subunit, and a fast-growing plus end, at which the β-tubulin subunit is exposed [2, 3]. MT assembly occurs in two phases: nucleation, which is facilitated by a third tubulin isoform, γ-tubulin; and elongation, during which αβ-tubulin heterodimers are added to the plus end [1, 4]. Tubulin is a unique guanine nucleotide-binding protein containing one exchangeable binding site and one nonexchangeable binding site. GTP at both sites is needed for optimal assembly, and GTP at the exchangeable site is hydrolyzed after assembly [5, 6]. This hydrolysis creates an MT consisting largely of GDP-tubulin; however, a small region of GTP-bound tubulin, called a “GTP cap,” remains at the end. This cap allows MTs to polymerize. The loss of the cap results in a transition from growth to shortening (called a “catastrophe”), whereas the reacquisition of the GTP cap results in a transition from shortening to growing (called a “rescue”). This behavior, known as dynamic instability, allows MTs to be remodeled rapidly in cells. An important consequence of dynamic instability is that it allows microtubules to search for specific target sites within the cell more effectively [7–9]. The MT assembly process is depicted in Figure 1.
MT assembly and stability can be affected by a wide variety of proteins. In this regard, microtubule-associated proteins (MAPs) play a very important role. Members of this group of proteins, such as MAP2 and tau, are known to promote MT assembly and stabilize MTs
Over the past decades, an effort has been made to understand the regulation of MT assembly and dynamics by signal transducing G proteins, as reviewed in Refs. [24, 25]. G proteins are heterotrimer, consisting of guanine nucleotide-binding α plus βγ subunits. The G-protein-signaling cascade begins with the agonist-induced activation of a G protein-coupled receptor (GPCR), which allows GTP to bind to the α subunit of the heterotrimer, and subsequently, the GTP-bound-activated Gα changes its association with Gβγ in a manner that permits both subunits to participate in the regulation of intracellular effector molecules [26]. The traditional pathway for GPCR signaling is shown in Figure 2. The GPCR family of proteins is highly diverse; more than 1000 gene-encoding GPCRs are found in the human genome [27, 28]. GPCRs participate in the regulation of a wide variety of physiological functions, including cell growth and differentiation, neurotransmission, immune system function, and hormonal signaling. Participation in such a multitude of processes makes GPCRs a very attractive drug target, and approximately 30% of commercially available drugs are designed to target GPCRs [29]. GPCRs consist of seven transmembrane domains, connected by three extracellular loops and three intracellular loops. The extracellular region is responsible for agonist binding (neurotransmitters, hormones, and odorants, among others), and the intracellular region is responsible for interacting with heterotrimeric G proteins [30]. In humans, there are 21 isoforms of Gα subunits, 6 Gβ isoforms, and 12 isoforms of Gγ [31]. G-protein heterotrimers are typically classified into four classes depending on the Gα subunit: Gαs (for stimulation of adenylyl cyclase), Gαi (for inhibition of adenylyl cyclase), Gαq (which regulates phospholipase), and Gα12/13, which is involved in the regulation of monomeric G proteins and other molecules, such as PKC [31, 32]. Typical effectors of Gα signaling include adenylyl cyclase, phospholipase C, phospholipase A2, ion channels, and several kinases and transcription factors. Termination of the signal occurs when GTP bound to the α subunit is hydrolyzed by its intrinsic GTPase activity that causes its functional dissociation from the effector and reassociation with βγ [26, 33–35]. Thus, G proteins act as molecular switches that can be turned “on” and “off” through the GTPase cycle. While the signal-transducing ability of heterotrimeric G proteins was once believed to depend fully on the α subunit, it has now become clear that the βγ subunit is capable of interacting with numerous effector molecules to influence a variety of signaling pathways [36, 37]. Among the effector molecules interacting with Gβγ are phospholipases, K+ and Ca2+ channels, GPCR kinases, members of the MAP kinase signaling pathway, monomeric G proteins, regulators of G protein signaling (RGS), and phosphoinositide-3 kinase (PI3K) [37–42].
Although G proteins are likely to be membrane-bound when coupled to receptors, results from several laboratories in past decades demonstrate their association with several subcellular compartments including MTs. G protein-MT interactions have been shown to modulate the assembly, dynamics and functions of MTs (Figure 2). This chapter focuses on our current understanding of G protein regulation of MT assembly and cellular and physiological aspects of this regulation.
2. Heterotrimeric G proteins and the tubulin/MT system
To investigate the potential link between Gβγ and MT assembly
3. G protein-microtubule interactions and cell division
Microtubules play a key role in cell division, participating in the exact organization and function of the spindle apparatus, a vehicle necessary for chromosomal segregation. Microtubules in the spindle are organized in such a way that the minus ends are near the spindle poles, while the plus ends extend toward the cell cortex or chromosomes [63]. Both α and βγ subunits of G proteins Gi and Go are consistently found to be associated with mitotic spindle. Genetic studies in
4. G protein-microtubule interactions and neuronal differentiation
The process by which MT structure is remodeled in neurons is a central question in cell biology and recent research indicates an important role of G protein subunits in this process. During neuronal differentiation, two distinct domains emerge from the cell body: a long, thin axon that transmits signals, and multiple shorter dendrites, which are specialized primarily for receiving signals. The axon terminal contains synapses, specialized structures where neurotransmitters are released to communicate with target neurons. Cytoskeletal structures embodied within neurite extensions and growth cone formations are essential for establishing appropriate synaptic connections and signal transmission. MTs form dense parallel arrays in axons and dendrites that are required for the growth and maintenance of such neurites. In the axon, MTs are bundled by tau, a microtubule-associated protein (MAP), with their plus end oriented toward the nerve terminal. MAP2, a group of high molecular weight MAPs, participates in MT bundling in the dendrites (Figure 3). Unlike MTs, actin filaments in neurons are enriched in growth cones and organized into long bundles that form filamentous protrusions, or filopodia, veil-like sheets of branched actin that form lamellipodia [1, 7, 73]. The interaction between these two cytoskeletal filaments is important for the advancement of growth cones and axon guidance [74, 75].
It is clear that cytoskeletal components can detect biochemical signals and respond in order to change the neuronal cell morphology. However, the precise signaling pathways that lead unique organization of MTs in neurons are not clearly understood [76]. PC12 cells have been used extensively for these studies as they respond to nerve growth factor (NGF) with growth arrest and exhibit a typical phenotype of neuronal cells that send out neurites [77]. NGF is a neurotrophic factor critical for the survival and maintenance of sensory and sympathetic neurons. The receptor commonly associated with this process is tyrosine kinase (TrkA) through which NGF exerts its effect [78]. PI3K appears to be the key molecule in this pathway and regulates localized assembly of MTs/actin filaments by downstream Akt/GSK3β pathways [79, 80]. The Rho and Ras families of small GTPases have also emerged as critical players in regulating the actin and MT cytoskeleton by modulating downstream effectors, including serine/threonine kinase, p21-activated kinase, ROCK, and mDia [81, 82]. GPCRs, as well as α and βγ subunits of heterotrimeric G proteins, have also been shown to regulate neurite outgrowth [83–90]. These studies collectively suggested the role of α and βγ subunits of G proteins in regulating neurite outgrowth. More recently, it has been demonstrated that both α and βγ subunits of G proteins regulate neurite outgrowth and differentiation by interacting with MTs and by modulating MT assembly/dynamics [24].
More recently, using biochemical and immunofluorescence analysis, it has been demonstrated that Gβγ-MT interactions and modulation of MT assembly is critical for NGF-induced neuronal differentiation of PC12 cells [94]. To address this, PC12 cells were treated with NGF over the course of three days to allow for neuronal differentiation. Microtubules (MTs) and soluble tubulin (ST) fractions were extracted using a microtubule-stabilizing buffer. The interaction of Gβγ with MT and ST fractions was analyzed by coimmunoprecipitating tubulin-Gβγ complex using a Gβ-specific antibody (rabbit polyclonal anti-Gβ) or a mouse monoclonal anti-α tubulin antibody and determining tubulin and Gβγ immunoreactivity in the complex [94]. Gβγ-MT interaction was significantly increased (2–3 fold) in NGF-treated cells. We also found that MT assembly was stimulated significantly (from 45.3 ± 4.8 to 70.1 ± 3.6%) in NGF-differentiated PC12 cells. The association of Gβγ with MTs in NGF-differentiated cells was also assessed by immunofluorescence microscopy [93]. After NGF treatment, the majority of the cells displayed neurite formation. Gβγ was detected in the neurites and in cell bodies. The colocalization of Gβγ with MTs/tubulin was observed along the neuronal process and in the central portion of the growth cone, but not at the tip of the growth cones.
Overexpression of Gβγ in PC12 cells induced neurite outgrowth in the absence of NGF, further supporting the role of Gβγ in neuronal differentiation [93]. Since Gβ1γ2 promoted MT assembly
Finally, the role of Gβγ in neuronal morphology, outgrowth and differentiation was further investigated using peptides and prenylation pathway inhibitors. For example, GRK2i, a Gβγ blocking peptide known to inhibit Gβγ-dependent effector functions, induced neurite damage as well as MTs and Gβγ aggregation. In addition, cellular aggregation was also frequently observed in the presence of GRK2i. The percentage of cell-bearing neurites was reduced significantly. On the other hand, synthetic peptide mSIRK, which is known to activate Gβγ signaling in cells by promoting the dissociation of Gβγ from α subunits, stimulated neurite formation. Since, γ-subunit of Gβγ is known to be posttranslationally modified by prenyl lipid, and prenylation deficient mutant of Gβγ (C68S) was shown to be functionally inactive, inhibitors of an enzyme of prenylation pathway (PMPMEase) was tested for their effects on MT assembly and neurite outgrowth. These inhibitors were found to alter MT organization and blocked neurite outgrowth. The results further demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and regulating MT assembly and organization.
5. Conclusion
Heterotrimeric G proteins transduce signals from cell surface receptors (G protein-coupled receptors) to intracellular effector molecules that include adenylyl cyclase, phospholipases, and ion channels. New evidence suggests that the modulation of the MTs by G proteins is an emerging field of research and therefore an in-depth understanding of G-protein-MTs interaction is important for elucidation of the function, behavior, and morphology of mammalian cells. Key results of this unique interaction may have a broader impact on health and diseases including cancer, Alzheimer’s, Parkinson’s, depression, and addictive behavior. We foresee that the G-protein-MT dependent pathway could be exploited for developing novel drugs to combat such diseases in the future.
Acknowledgments
Research in author’s laboratory described in this report was, in part, supported by G12MD007592 (NIHMHD) to the University of Texas at El Paso. An R01 subcontract to SR from the University of Illinois at Chicago (MH39595) supported some earlier research in author’s laboratory. The biochemical, molecular, and confocal microscopy experiments were carried out at the Biomolecule Analysis Core Facility, Genome Analysis Core facility, and Cytometry/screening/imaging Facility at the Border Biomedical Research Center (UTEP) supported by a grant (G12MD007592) from NIMHD (NIH). The authors like to thank Dr. Siddhartha Das for critically reading the manuscript and thoughtful suggestions.
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