Mutational analysis of conserved amino acids in the T cell receptor alpha-chain transmembrane region: a critical role of leucine 112 and phenylalanine 127 for assembly and surface expression.
Correct assembly of all TCR complex polypeptides is essential for its cell surface expression and function. The transmembrane region of the TCRalpha chain is highly conserved and to gain insight into the structural and functional role of these residues, single amino acid substitutions were introduced and surface expression and signaling ability studied in T hybridoma cells. Introduction of acid residues within the TCRalpha chain transmembrane region were mostly tolerated, indicating that the net charge within this region of the TCR complex is not crucial to either assembly or signaling. However, mutations of leucine 112 or phenylalanine 127 to aspartic acids (L112D or F127D, respectively) resulted in dramatic loss of surface expression and, therefore, their signaling ability. Intracellular flow cytometry showed that the mutant TCRalpha polypeptides were present at levels comparable to wild-type, indicating that the reduced surface expression was not a consequence of impaired protein survival. The defect was characterized by immunoprecipitation and showed that residues L112 and F127 were involved in early interactions with the CD3 complex. A large proportion of the TCRalpha chain mutants L112D and F127D consisted of immature protein, indicative of a problem during early assembly of the TCR. Our findings provide evidence for the involvement of the conserved L112 and F127 residues of the TCRalpha chain transmembrane region in the assembly process of the TCR complex.
Bhatnagar
Aparna
A
Malaghan Institute of Medical Research, P.O. Box 7060, Wellington South, New Zealand.
Gülland
Sven
S
Bascand
Micaela
M
Palmer
Ed
E
Gardner
Terrence G
TG
Kearse
Kelly P
KP
Bäckström
B Thomas
BT
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D006801:Humans; D006825:Hybridomas; D007930:Leucine / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000502:physiology*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D000596:Amino Acids; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; C054979:antigen T cell receptor, zeta chain; D010649:Phenylalanine; D007930:Leucine