Ch_6/7

G proteins are formed of an alpha (40-45 kDa), ß (35-36 kDa) and y (8-18 kDa) SU.

The alpha SU is involved in receptor recognition, effector recognition, RGS protein interactions and possesses GTPase activity.

The ßy SU is involved in GDP-bound Galpha SU recognition, receptor recognition and effector recognition.

 The G protein cycle: an activated GPCR functions as a GEF, facilitating GDP for GTP exchange on the Galpha SU of the G protein. GTP-bound Galpha dissociates from the ßy SUs and the two complexes are thus free to regulate effectors. Signalling is terminated by hydrolysis of the GTP bound to Galpha. The GDP-bound Galpha SU then re-associates with Gßy and signalling is terminated.

Galpha-GTP and Gßy SUs transduce their signals to e.g.: AC (ATP -> cAMP +PPi); PLC (PIP2 -> IP3 + DAG); PI3K (PIP2 -> PIP3). Ion channels are also modulated e.g. P/Q,T-type & N-type VOCCs and GIRKs (G proteins inwardly-rectifying K+ channels).

Effectors like AC and PLC are primarily regulated by the Galpha SU. Effectors such as P/Q & T-type VOCCs and GIRKs are regulated by the ßy SU of the G proteins. PLC can be activated by ßy. Some ACs activated by ßy. L-type VOCCs activated by Gas and not at all by ßy.

Galpha subfamilies include Gs, Gi/o, Gq/11 & G12/13. Gß subfamilies: Gß1-5. Gy subfamilies: Gy1-13. The N-terminus of Galpha is lipidated for anchoring to the PM. The C-terminus of the y SU is lipidated for PM anchoring (allows concentration of the proteins in 2D on the membrane. The ß SU associates covalently with the y SU via a coiled-coil domain. 

The range of alpha, ß and y SUs could, in theory, give rise to ~1000 different combinations. Generally, Gs alpha SUs upregulate adenylyl cyclase (AC) activity; Gi/o are involved in ion channel modulation and reduction in AC activity; Gq/11  are involved in PLC activation; G12/13 (12 & 13) are involved in scaffolding regulators of small G proteins.

The specificity of receptor-G protein interactions lies in the C terminal tail of the GPCR. For example, changing the 5 C-terminal residues of a Gq-coupled GPCR from EYNLV to that of a Gi coupled GPCR e.g. DCGLF changes the receptor specificity i.e. the chimeric GPCR will now interact with Gi rather than Gq.

- Use of: AlF4- (alluminium fluoride) which mimics GTP (due to -ve charge like phosphate) and is used as a general activator of G proteins.

- GTPyS  a non-hydrolysable GTP analog which is a persistent activator of Galpha.

- Bacterial toxins can also modify G proteins. Cholera toxin ADP-ribosylates the Gas SU such that it can no longer hydrolyse GTP. Thus, the G protein is constitutively active, and cAMP levels greatly increase. Pertussis toxin ADP-ribosylates Gai such that it can no longer exchange GDP for GTP, turning off its activity (cAMP increases again as inhibition of AC by Gi relieved).

- Antibodies can also be used to block receptor-G protein interactions for study. Immunoprecipitation of particular Galpha SUs can be used to characterise G protein-receptor coupling.

Defining a population of G proteins activates by a receptor: add radiolabelled GTPyS to a cell such that it will bind to G proteins undergoing activation. Addition of agonist to the receptors activates them to facilitate GDP for GTP exchange on a subpopulation of Galpha SUs which will contain GTPyS rather than GTP. Next, Ab are used to immunoprecipitate out G alpha SUs. A scintillation counter is used to detect co-immunoprecipitated GTPyS radioactivity.

The intrinsic GTPase activity of Galpha is generally insufficient to account for the Koff rate of G proteins (the rate at which GTP bound to Galpha is hydrolysed to GDP to terminate signalling). Thus, co-factors must be involved. Originally, it was thought that interactions with effectors e.g. cGMP PDE or PLCß could modulate GTPase activity. However, it is now known that a family of GTPase activating proteins (GAPs) function to accelerate the GTPase activity of Galpha. GAPs are regulators of G protein signalling (RGS)

~30 RGS proteins are now known. They possess RGS domains which are ~120 residues long and increase GTPase activity on a subset of Galpha SUs. Some proteins possess RGS-like domains which bind to Galpha but don’t increase GTPase activity

The G protein cycle is regulated by GEFs which facilitate GDP for GTP exchange (e.g. GPCRs), guanine nucleotide dissociation inhibitors (GDI) which bind Galpha to prevent signalling (e.g. ßy) and GAPs which turn off signalling by facilitating hydrolysis of GTP bound to Galpha.

Activation of M2 mAChRs by ACh activates Gßy which functions here to activate GIRKs. Expressing GIRKs and mAChRs in CHO cells and activating by using ACh results in a much more sustained response than that seen in vivo in atrial cells (10.9 s to deactivate in contrast to 0.6 s in vivo). Upon co-expression with RGS9, the time course of activation then resembles that in the heart (0.8 s).a

This can also be seen in the mouse photoreceptor. Time to recovery from photobleaching in RGS9-1 KO mice is markedly delayed compared to heterozygotes (+/-) and homozygotes (+/+) - these two show similar kinetics suggesting the protein is in excess.

RGS proteins not only show selectivity towards Galpha SUs but also toward receptors. Pancreatic acinar cells express M3, CCK1 and BB1 receptors, all of which couple to Gq (lead to Ca2+ release from stores). Muallem et al. assessed the differential effects of RGS4 on signalling from these three receptors.

Muallem et al. varied the overexpression of RGS4 in cells with these receptors and assessed the level of inhibition of Ca2+-release (i.e. more RGS4 more inhibition of release due to Gq signalling termination). M3 found to be most sensitive (curve most to the left), then BB1 and CCK1. These results illustrate differential sensitivity, and as they use the same G proteins, this difference in sensitivity to RGS9-1 seems to lie at the level of the receptor i.e. the receptor influences this specificity.