Abstract
It is well established that several cell surface receptors interact with each other to form dimers and oligomers, which are essential for their activation. Since little is known about the quaternary structure of P2Y receptors, in the present work, we investigated the expression of the G-protein-coupled P2Y&sub4; subunit as monomeric or higher-order complex protein. We examined both endogenously expressed P2Y&sub4; subtype with the aid of specific anti-P2Y&sub4; antiserum, and heterologously transfected P2Y&sub4;-tagged receptors with the use of antitag antibodies. In both cases, we found the P2Y&sub4; receptor displaying molecular masses corresponding to monomeric, dimeric and oligomeric structures. Experiments performed in the absence of reducing agents demonstrated that there is a strict correlation among the multiple protein bands and that the multimeric forms are at least partially assembled by disulphide bonds. The direct demonstration of P2Y&sub4; homodimerisation comes instead from co-transfection and differential co-immunoprecipitation experiments, with the use of differently tagged P2Y&sub4; receptors and antitag antibodies. The structural propensity of the P2Y&sub4; protein to form homo-oligomers may open the possibility of a novel regulatory mechanism of physiopathological functions for this and additional P2Y receptors.