Role of Amino- and Carboxyl-Terminal Regions of G_αzin the Recognition of G_i-Coupled Receptors

Abstract

Many Gi-coupled receptors are known to interact with the pertussis toxin (PTX)-insensitive Gz protein. Given that the alpha subunits of Gi and Gz share only 60% identity in their amino acid sequences, their receptor-interacting domains must be highly similar. By swapping the carboxyl termini of alpha i2 and alpha z with each other or with those of alpha t, alpha12, and alpha13, we examined the relative contributions of the carboxyl-end 36 amino acids of the alpha chains toward receptor recognition. Chimeric alpha chains lacking the site for PTX-catalyzed ADP-ribosylation were coexpressed with the type II adenylyl cyclase (AC II) and one of several Gi-coupled receptors (formyl peptide, dopamine D2, and delta-opioid receptors) in human embryonic kidney 293 cells. The alpha i2/alpha z chimera was able to interact with both aminergic and peptidergic receptors, resulting in betagamma-mediated stimulation of AC II in the presence of agonists and PTX. Functional and mutational analyses of alpha i2/alpha z revealed that this chimera can inhibit the endogenous ACs of 293 cells. Similarly, the alpha z/alpha i2 chimera seemed to retain the abilities to interact with receptors and inhibit cAMP accumulation. Fusion of the carboxyl-terminal 36 amino acids of alpha z to a backbone of alpha t1 produced a chimera, alpha t1/alpha z, that did not couple to any of the Gi-coupled receptors tested. Interestingly, an alpha13/alpha z chimera (with only the last five amino acids switched) displayed differential abilities to interact with receptors. Signals from aminergic, but not peptidergic, receptors were transduced by alpha13/alpha z. A similar construct, alpha12/alpha z, behaved just like alpha13/alpha z. These results indicated that "alpha i-like" or "alpha z-like" sequences at the carboxyl termini of alpha subunits are not always necessary or sufficient for specifying interaction with Gi-coupled receptors.

Gα 12 - and Gα 13 -Protein Subunit Linkage of D 5 Dopamine Receptors in the Nephron

“…27,28 HEK293 cells also express G␣ 13 , 27 although to a lesser degree than G␣ 12 , which may have led earlier reports to conclude that G␣ 13 is not endogenously expressed in this cell line. 28 Like the results observed with renal proximal tubule cells, D 5 colocalized with G␣ 12 , and G␣ 13 , which was also increased by fenoldopam ( Figures 3D and 3E). No such colocalization was found in HEK293 cells transfected with the empty vector (not shown).…”

Gz signaling: emerging divergence from Gi signaling

“…However, Ga t1 shows no coupling to most of the G i -linked receptors. Swapping the last 36 amino acids of Ga t1 with those derived from Ga z could not redirect the chimera to interact with G i -coupled receptors (Tsu et al, 1997), thus indicating that the Cterminus of Ga z is not the only essential region specifying its receptor coupling speci®city. An extensive chimera study utilizing Ga z and Ga t1 as parental Ga subunits has identi®ed other essential elements for ecient receptor coupling by Ga z .…”

“…These include the adenosine A 1 , a 2 -adrenergic, dopamine D 2 (Wong et al, 1992), 5-HT 1A (Butkerait et al, 1995), muscarinic M 2 (Parker et al, 1991), substance P (Barr et al, 1997), and all types of opioid receptors (Chan et al, , 1998Lai et al, 1995;Tsu et al, 1995a). In addition, G z is capable of transducing signals from chemoattractant receptors such as the complement C5a (Shum et al, 1995) and formyl peptide (Tsu et al, 1995b) receptors. The overall receptor recognition pro®le of G z initially appeared to be identical to those of the G i proteins because, without exception, every receptor that coupled to G z also recognized G i subunits.…”

Gα16/z Chimeras Efficiently Link a Wide Range of G Protein–Coupled Receptors to Calcium Mobilization

“…19 Likewise, G i -linked receptors require the N-terminus of Gα z for efficient coupling. 20,21 Although the Gα q chimeras with modifications at the N-or C-terminus exhibit higher promiscuity for GPCRs than the parental Gα q , Gα 16 remains more promiscuous than the Gα q chimeras. 22 We have recently embarked on a quest to generate universal G protein adapters by using Gα 16 as a backbone.…”