Regulation of G Protein Activation and Effector Modulation by Fusion Proteins between the Human 5-Hydroxytryptamine1A Receptor and the a Subunit of Gi1a: Differences in Receptor- Constitutive Activity Imparted by Single Amino Acid Substitutions in Gi1a

Fusion proteins were generated between the human 5-hydroxytryptamine (5-HT)1A receptor and both wild-type (Cys ) and pertussis toxin-resistant (Gly and Ile) forms of Gi1. These were expressed stably. Pertussis toxin treatment substantially reduced basal high-affinity GTPase activity in clones expressing the 5-HT1A receptor wild-type Gi1a construct but not in clones expressing 5-HT1A receptor (Gly )Gi1a or (Ile)Gi1a. Spiperone functioned as an inverse agonist in membranes expressing the 5-HT1A receptor wild-type Gi1a fusion protein and in those expressing 5-HT1A receptor (Ile)Gi1a but not the 5-HT1A receptor (Gly )Gi1a fusion protein. The effect of spiperone at the 5-HT1A receptor wild-type Gi1a construct but not the 5-HT1A receptor (Ile )Gi1a construct was blocked by pertussis toxin treatment. By contrast, agonists functioned with equal effectiveness at the three fusion proteins and were unaffected by pertussis toxin treatment of the (Ile)Gi1aand (Gly )Gi1a-containing constructs. 5-HT resulted in strong inhibition of forskolin-amplified adenylyl cyclase in intact cells expressing the isolated 5-HT1A receptor. In fusion protein-expressing cells, 5-HT-mediated inhibition of adenylyl cyclase was also observed. Pertussis toxin treatment obliterated 5-HT-mediated inhibition in cells expressing the isolated receptor and the 5-HT1A receptor wild-type Gi1a fusion protein but not in those expressing the 5-HT1A receptor (Ile ) or (Gly)Gi1a fusion proteins. These studies demonstrate that alteration of a single amino acid in Gi1a located at a key contact site between the G protein and a G protein-coupled receptor can regulate agonist-independent constitutive activity of the G protein-coupled receptor and that fusion proteins can directly regulate adenylyl cyclase. 5-hydroxytryptamine (5-HT) mediates a wide range of physiological actions via activation of a large family of receptors. With the exception of the 5-HT3 receptor, which is an intrinsic cation channel, all of the receptors for 5-HT are members of the superfamily of seven transmembrane element, G protein-coupled receptors (GPCRs). A highly studied member of this family is the 5-HT1A receptor, which is expressed both presynaptically on serotinergic nerve bodies, where it functions as an autoreceptor to dampen neuronal activity, and postsynaptically in many locations in the central nervous system to which serotinergic neurones project. Signal transduction from this GPCR is mediated predominantly via activation of members of the pertussis toxin-sensitive family of Gi-like G proteins and involves inhibitory regulation of adenylyl cyclase as well as modulation of the activity of a series of ion channels (Julius, 1998). Considerable interest has been accorded pharmacological studies indicating a likely role for this GPCR in the regulation of anxiety states, and the recent production of mice lacking expression of this GPCR has confirmed these ideas (Heisler et al., 1998; Ramboz et al., 1998). In a series of studies designed to address details of the interactions of GPCRs and G proteins, we have recently constructed a number of fusion proteins in which the N terminus of a G protein a subunit was linked directly to the C-terminal tail of a GPCR (Wise and Milligan, 1997; Wise et al., 1997a,b, 1999). Some of the constructs we have examined in detail have utilized the a2A adrenoceptor (Wise and Milligan, 1997; Wise et al. 1997a,b). This GPCR is well estabThis work was supported by the Medical Research Council and the European Union Biomed II program: Inverse agonism: Implications for drug design. ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; GPCR, G protein-coupled receptor; MPPF, 4(29-methoxy)-phenyl-1-[29-(N-20-pyridinyl)-p-fluorobenzamido]ethyl-piperazine; OH-DPAT, hydroxy-2-(di-n-propylamino)tetralin; PCR, polymerase chain reaction; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis. 0026-895X/99/040684-09$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 56:684–692 (1999). 684 at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from lished to function predominantly via members of the Gi family of G proteins. After stable expression of an a2A adrenoceptor-Gi1a fusion protein in which the G protein was altered by conversion of cysteine to glycine to render it insensitive to the actions of pertussis toxin, we noted that although inhibition of adenylyl cyclase could be produced by a2A adrenoceptor agonists in untreated cells, this was abolished after pertussis toxin treatment and thus could not have been mediated via the G protein linked to the GPCR (Burt et al., 1998). In the present study, we have generated fusion proteins between the human 5-HT1A receptor and both wild-type (Cys) and pertussis toxin-insensitive (Gly and Ile) forms of Gi1a. After stable expression of these constructs in human embryonic kidney (HEK)293 cells, ligand-mediated modulation of their GTPase activity and regulation of adenylyl cyclase activity were examined. As point mutation of residue of Gi1a can alter both the maximal effectiveness of agonist ligands (Bahia et al., 1998; Carr et al., 1998) and the relative intrinsic activity of different agonists (Jackson et al., 1999) we wished to assess how agonist-independent, constitutive activity might be modified by mutation of this residue, which lies within a key GPCR-G protein contact domain. We note marked constitutive activity of the 5-HT1A receptor wild-type Gi1a fusion protein and the 5-HT1A receptor (Ile)-Gi1a fusion protein, but not the 5-HT1A receptor (Gly)-Gi1a fusion protein, which could be inhibited by the inverse agonist spiperone. Confirmation that the constitutive activity of the 5-HT1A receptor (Ile ) Gi1a fusion protein was inherently derived from intramolecular interactions of the two elements of the fusion protein was provided by the inability of pertussis toxin treatment to prevent spiperonemediated inhibition of GTPase activity at this construct. These studies provide the first demonstration that single amino acid alterations in a G protein can significantly alter agonist-independent constitutive activity of a GPCR. Furthermore, we also record clear agonist-induced inhibitory regulation of adenylyl cyclase activity, which proceeds via the G protein of the fusion constructs. Experimental Procedures Materials. All materials for tissue culture were supplied by Life Technologies Inc. (Paisley, Strathclyde, UK). Both the 5-HT1A receptor antagonist [H]4(29-methoxy)-phenyl-1-[29-(N-20-pyridinyl)-p-fluorobenzamido]ethyl-piperazine (MPPF; 78.3 Ci/mmol) and [g-P]GTP (30 Ci/mmol) were obtained from DuPont-NEN (Boston, MA). Cholera toxin and pertussis toxin were purchased from Sigma (St. Louis, MO). Oligonucleotides were purchased from Cruachem (Glasgow, Strathclyde, UK). All other chemicals were obtained from Sigma and Boehringer Mannheim (Mannheim, Germany). Construction of Plasmids Encoding 5HT1A, 5HT1A-Gi1a Fusion Proteins. The human 5HT1A receptor clone in pSP64 (a gift from Glaxo-Wellcome, Stevenage, UK) was digested with XbaI/ BamHI and the resulting 1.5-kb fragment ligated to pcDNA3. To obtain the open reading frame of 1.3 kb, polymerase chain reaction (PCR) was carried out using the following primers to introduce a HindIII restriction site at the 59 end and to remove the stop codon and introduce a BamHI restriction site at the 39 end, respectively: 59-CTGAAGCTTATGGATGTGCTCAGCCCTGGTC-39; 59-CTGGGA TCCCTGGCGGCAGAAGTTACACTTAATG-39 (restriction enzyme sites underlined). The PCR fragment was digested with HindIII and BamHI and ligated into pcDNA3 to make the plasmid p5HT. To link the Gi1a wild-type (Cys ) cDNA to the 5HT1A receptor sequence, PCR was carried out on Gi1a to produce compatible restriction sites. The oligonucleotides used to do this were 59-CTGGGATCCGGCTGCACACTGAGCGCTGAG-39 at the 59 end and 59-GAGAATTCTTAGAAAGAGACCACAGTC-39 for the 39 end. The plasmid p5HT was digested with BamHI/EcoRI as was the Gi1a PCR fragment and the two were ligated to give the plasmid p5HTGi1. To construct the 5HT1A-(Gly )Gi1a fusion plasmid (Gly )Gi1a in PBS was digested with SacII/EcoRI and the 730-bp fragment was used to replace the corresponding fragment in p5HTGi1. An equivalent strategy was used to produce 5HT1A-(Ile )Gi1a. The constructs were then sequenced to verify the DNA sequence. Cell Culture and Stable Expression. HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) newborn calf serum, and 2 mM L-glutamine. Cells were seeded into 100-mm culture dishes and grown to 60 to 80% confluence (18–24 h) before transfection with 5 mg of appropriate cDNAs using N-[1-(2,3-dideoxyloxy)propyl]-N,N,N-trimethyl ammonium methyl sulphate reagent (Boehringer Mannheim). Forty-eight hours after transfection, the cells were split 1:4 into 800 mg/ml G418 sulfate (Calbiochem, La Jolla, CA) containing medium. A 100-mm dish of untransfected HEK293 cells was also split into the same medium as a control dish. About 1 week later, after all the cells in the control dish had died, G418-resistant cells in the transfected dishes were picked and transferred into 24-well plates using autoclaved pipette tips. About 20 clones of each cDNA were picked and grown in 1 ml/well of G418 medium (400 mg/ml). Clones were amplified, membrane preparations made, and their binding of [H]MPPF deter-