Identification of a Polar Region in Transmembrane Domain 6 That Regulates the Function of the G Protein-Coupled α-Factor Receptor

ABSTRACT The α-factor pheromone receptor (Ste2p) of the yeastSaccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the α-factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the α-factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser254 showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln253 and Ser254 are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln253 and Ser254 fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln253and Ser254 face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser288 and Ser292) that are potential contact residues for Gln253 because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the α-factor receptor that regulates its ability to enter the activated conformation.

Download Full-text

The Cytoplasmic End of Transmembrane Domain 3 Regulates the Activity of the Saccharomyces cerevisiae G-Protein-Coupled α-Factor Receptor

Genetics ◽

10.1093/genetics/160.2.429 ◽

2002 ◽

Vol 160 (2) ◽

pp. 429-443

Author(s):

William Parrish ◽

Markus Eilers ◽

Weiwen Ying ◽

James B Konopka

Keyword(s):

G Protein ◽

Transmembrane Domain ◽

Transmembrane Helix ◽

G Protein Coupled Receptors ◽

Transmembrane Domains ◽

Targeted Mutagenesis ◽

Activating Mutations ◽

Polar Residues ◽

Domain 3 ◽

G Protein Coupled

Abstract The binding of α-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the α-factor receptor that includes transmembrane domains 1–5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the α-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors.

Download Full-text

Involvement of the constitutive activity of the GHS-R1a (ghrelin G-protein coupled receptor) in the tumorigenesis of somatotroph adenomas

Endocrine Abstracts ◽

10.1530/endoabs.32.oc1.2 ◽

2013 ◽

Author(s):

Yves Louis Mear ◽

Xavier Come Donato ◽

Marie Pierre Blanchard ◽

Celine Defilles ◽

Christophe Lisbonis ◽

...

Keyword(s):

G Protein ◽

Constitutive Activity ◽

G Protein Coupled Receptor ◽

G Protein Coupled

Download Full-text

Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

PLoS ONE ◽

10.1371/journal.pone.0138463 ◽

2015 ◽

Vol 10 (9) ◽

pp. e0138463 ◽

Cited By ~ 53

Author(s):

Adam L. Martin ◽

Michael A. Steurer ◽

Robert S. Aronstam

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Constitutive Activity ◽

Class A ◽

G Protein Coupled

Download Full-text

Solution structure of the sixth transmembrane helix of the G-protein-coupled receptor, rhodopsin11This work was supported by National Institutes of Health Grant EY03328 and in part by CA16056.

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/s0005-2736(99)00212-6 ◽

2000 ◽

Vol 1463 (1) ◽

pp. 1-5 ◽

Cited By ~ 28

Author(s):

Atin Chopra ◽

Philip L. Yeagle ◽

James A. Alderfer ◽

Arlene D. Albert

Keyword(s):

G Protein ◽

Solution Structure ◽

Transmembrane Helix ◽

National Institutes Of Health ◽

G Protein Coupled Receptor ◽

G Protein Coupled

Download Full-text

G-protein-coupled-receptor activation of the smooth muscle calponin gene

Biochemical Journal ◽

10.1042/bj3570587 ◽

2001 ◽

Vol 357 (2) ◽

pp. 587-592 ◽

Cited By ~ 9

Author(s):

Nickolai O. DULIN ◽

Sergei N. ORLOV ◽

Chad M. KITCHEN ◽

Tatyana A. VOYNO-YASENETSKAYA ◽

Joseph M. MIANO

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

G Protein ◽

Transcriptional Activation ◽

Fold Increase ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Mitogen Activated Protein Kinase ◽

G Protein Coupled

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [α-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.

Download Full-text

Trans-Activation of Mutant Follicle-Stimulating Hormone Receptors Selectively Generates Only One of Two Hormone Signals

Molecular Endocrinology ◽

10.1210/me.2003-0443 ◽

2004 ◽

Vol 18 (4) ◽

pp. 968-978 ◽

Cited By ~ 40

Author(s):

Inhae Ji ◽

ChangWoo Lee ◽

MyoungKun Jeoung ◽

YongBum Koo ◽

Gail A. Sievert ◽

...

Keyword(s):

Adenylyl Cyclase ◽

G Protein ◽

Hormone Receptors ◽

Transmembrane Domain ◽

Signal Generation ◽

Lh Receptor ◽

Glycosyl Phosphatidylinositol ◽

Mutant Receptors ◽

G Protein Coupled ◽

Hormone Signals

Abstract Previously, we reported that a liganded LH receptor (LHR) is capable of activating itself (cis-activation) and other nonliganded LHRs to induce cAMP (trans-activation). Trans-activation of the LHR raises two crucial questions. Is trans-activation unique to LHR or common to other G protein-coupled receptors? Does trans-activation stimulate phospholipase Cβ as it does adenylyl cyclase? To address these questions, two types of novel FSH receptors (FSHRs) were constructed, one defective in hormone binding and the other defective in signal generation. The FSHR, a G protein-coupled receptor, comprises two major domains, the N-terminal extracellular exodomain that binds the hormone and the membrane-associated endodomain that generates the hormone signals. For signal defective receptors, the exodomain was attached to glycosyl phosphatidylinositol (ExoGPI) or the transmembrane domain of CD8 immune receptor (ExoCD). ExoGPI and ExoCD can trans-activate another nonliganded FSH. Surprisingly, the trans-activation generates a signal to activate either adenylyl cyclase or phospholipase Cβ, but not both. These results indicate that trans-activation in these mutant receptors is selective and limited in signal generation, thus providing new approaches to investigating the generation of different hormone signals and a novel means to selectively generate a particular hormone signal. Our data also suggest that the FSHR’s exodomain could not trans-activate LHR.

Download Full-text