Although initial studies showed alterations in adenylyl cyclase activity in response to microtubule-disrupting agents, it remained unclear whether this was due to a direct interaction between Gsα and tubulin, or the result of disruption of cellular architecture [41
]. In vitro, and in permeabilized cells, tubulin-GPPNHP (tubulin covalently liganded to a non-hydrolyzable GTP analogue) activates Gsα independently of receptor [64
]. However, this has not been seen in living cells. Rather, microtubule-disrupting agents may affect a scaffolding or organizing role of membrane tubulin, which can alter the stability of G-protein-signaling complexes. Indeed, treatment of cardiomyocytes with colchicine causes AC activation and promotes translocation of Gsα and its effector adenylyl cyclase into similar membrane domains (non-lipid raft membrane fractions) [22
]. Similar effects were seen in S49 cells, which lack lipid rafts, suggesting that Gsα activation of AC occurs outside of these regions. In summary, alterations in cAMP formation due to microtubule-disrupting agents may be a result of alterations in cytoskeletal organization of the membrane rather than direct Gsα-tubulin interactions.
What are the functional consequences of direct Gα-tubulin interactions? Giα was observed to destabilize microtubules by stimulating the tubulin-GTP hydrolysis rate [47
]. This effect persisted even if a GTPase-deficient GiαQ204L
mutant was used to prevent Giα from hydrolyzing GTP on tubulin. This could not be reversed by addition of exogenous non-hydrolyzable GTP analogues. Consistent with the effect on tubulin GTPase activity, Giα affected microtubule dynamics by increasing the frequency of catastrophes (number of rapid microtubule shortening per second) without affecting rescue frequency or growing or shortening rate [47
]. This has the net effect of converting long microtubules into a greater number of shorter microtubules.
Gsα appears to work by a similar mechanism. In response to agonist, Gsα internalizes through caveolae/lipid raft-derived vesicles, thereby facilitating interaction with the plus ends of microtubules that are rich in tubulin-GTP [3
]. There, active Gsα stimulates hydrolysis of tubulin-GTP, and likely increases microtubule dynamics and decreases microtubule stability [31
]. Moreover, active Gsα may sequester newly released tubulin-GDP to prevent repolymerization. After some time, Gsα hydrolyzes its own GTP to GDP, adopts the inactive conformation, and releases tubulin. In cells, the result is an increase in process formation in response to Gα-activation [11
One issue that arises is why Gsα and Giα have similar, rather than opposing, effects on microtubule polymerization. One must keep in mind that the designations ‘stimulatory’ and ‘inhibitory’ are somewhat simplistic, and were generated to refer to the activities of these G-proteins on adenylyl cyclase. Indeed, Gsα- and Giα-mediated signaling pathways interact fruitfully with each other in a complex manner. For example, the β2
-AR can couple to both Gsα and Giα to regulate airway reactivity in asthma, and Gsα-coupled β-ARs can heterodimerize with Giα-coupled opioid receptors [69
]. Furthermore, while Giα and Gsα interact with different surfaces of adenylyl cyclase, the two proteins probably interact with a similar surface on tubulin [31
]. The presumed interface of both Giα and Gsα with tubulin on the G-protein includes the region between switch II and switch III, a region also involved in binding Gβγ and effectors such as adenylyl cyclase (fig. ). Finally, the effects of Gα subunits on tubulin are direct and independent of adenylyl cyclase.
Recent studies have revealed a role for Gβγ subunits in modulating microtubule polymerization as well. Gβ1
, but not Gβ1
or a prenylation-deficient Gβ1
mutant, promotes microtubule polymerization, both in vitro and in cells [40
]. This occurs even in the absence of microtubule associated proteins, suggesting a direct interaction between Gβγ and tubulin. Gqα-agonist stimulation of cells causes receptor, Gβγ, and tubulin (but not Gqα) to internalize in clathrin-coated vesicles [68
]. Once inside the cell, Gβγ binds along the length of microtubules (but not to dimeric tubulin) to increase microtubule stability [38
]. Giα also interferes with the ability of Gβγ to stabilize microtubules, as the latter protein is inactive when preincubated with heterotrimeric Giαβγ [46
]. Since the active tubulin-binding interfaces for Gα and Gβγ are probably occluded in the heterotrimer, the heterotrimer may bind tubulin via an alternate binding site on Gα or Gβγ [53
]. In conclusion, Gβγ and Gα subunits have opposite effects on microtubules through distinct mechanisms.
Gqα also binds to tubulin with 130 nM
affinity, but its effects on tubulin are very different from Gsα and Giα. Stimulation of Gqα-coupled receptors recruits tubulin to the membrane [12
]. This interaction involves GTP-tubulin, and occurs on a time course similar to PLC-β1
]. Microtubule stabilization appears to inhibit this process and microtubule depolymerization mimics it. Activation of Gqα-coupled mGluRs promotes microtubule depolymerization in cells. This may be due to Ca2+
released from intracellular stores as a result of IP3
Another relationship between G-proteins and cytoskeleton is the role of microtubule and actin filament on translocation of transducin or Gtα translocation in rod photoreceptor cells. Analogous to Gsα, Gtα undergoes a light (‘agonist’)-dependent translocation from the rod outer segment to the inner segment within minutes, and the reverse slowly occurs in the dark [9
]. The two segments of rods are connected by a non-motile cilium. Although initial studies proposed the translocation to occur via diffusion, the cytoskeleton also plays a role in this process [44
]. Gtα colocalizes with microtubules in dark-adapted retinas. Light-dependent translocation of Gtα did not depend on microfilaments (cytochalasin-D independent) or microtubules (thiabendazole treatment). In contrast, the reverse translocation of Gtα in the dark depends on both microfilaments and microtubules [57
]. Note, however, that Gtα does not bind tubulin or microtubules directly [60
]. Thus, the mechanisms of ‘agonist’-induced Gsα and Gtα translocation are likely divergent.